Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing succinate related thereto

ABSTRACT

Provided herein is a non-naturally occurring microbial organism having a methanol metabolic pathway that can enhance the availability of reducing equivalents in the presence of methanol. Such reducing equivalents can be used to increase the product yield of organic compounds produced by the microbial organism, such as succinate. Also provided herein are methods for using such an organism to produce succinate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Ser. No. 61/766,642 filedFeb. 19, 2013, and U.S. Ser. No. 61/717,001 filed Oct. 22, 2012, each ofwhich is incorporated herein by reference in its entirety.

1. SUMMARY

Provided herein are methods generally relating to metabolic andbiosynthetic processes and microbial organisms capable of producingorganic compounds. Specifically, provided herein is a non-naturallyoccurring microbial organism having a methanol metabolic pathway thatcan enhance the availability of reducing equivalents in the presence ofmethanol. Such reducing equivalents can be used to increase the productyield of organic compounds produced by the microbial organism, such assuccinate. Also provided herein are non-naturally occurring microbialorganisms and methods thereof to produce optimal yields of succinate.

In a first aspect, provided herein is a non-naturally occurringmicrobial organism having a methanol metabolic pathway, wherein saidorganism comprises at least one nucleic acid encoding a methanolmetabolic pathway enzyme expressed in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol. Incertain embodiments, the methanol metabolic pathway comprises one ormore enzymes selected from the group consisting of a methanolmethyltransferase; a methylenetetrahydrofolate reductase; amethylenetetrahydrofolate dehydrogenase; a methenyltetrahydrofolatecyclohydrolase; a formyltetrahydrofolate deformylase; aformyltetrahydrofolate synthetase; a formate hydrogen lyase; ahydrogenase; a formate dehydrogenase; a methanol dehydrogenase; aformaldehyde activating enzyme; a formaldehyde dehydrogenase; aS-(hydroxymethyl)glutathione synthase; a glutathione-dependentformaldehyde dehydrogenase; and an S-formylglutathione hydrolase. Suchorganisms advantageously allow for the production of reducingequivalents, which can then be used by the organism for the productionof succinate using any one of the succinate pathways provided herein.

In a second aspect, provided herein is a non-naturally occurringmicrobial organism having (1) a methanol metabolic pathway, wherein saidorganism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme expressed in a sufficient amount toenhance the availability of reducing equivalents in the presence ofmethanol; and (2) a succinate pathway. In some embodiments, the organismfurther comprises at least one nucleic acid encoding a succinate pathwayenzyme expressed in a sufficient amount to produce succinate. In someembodiments, the nucleic acid is an exogenous nucleic acid. In otherembodiments, the nucleic acid is an endogenous nucleic acid. In certainembodiments, the succinate pathway enzyme is selected from the groupconsisting of a phosphoenolpyruvate (PEP) carboxylase or a PEPcarboxykinase; a pyruvate carboxylase; a malate dehydrogenase; a malicenzyme; a fumarase; and a fumarate reductase.

In other embodiments, the organism having a methanol metabolic pathway,either alone or in combination with a succinate pathway, as providedherein, further comprises a formaldehyde assimilation pathway thatutilizes formaldehyde, e.g., obtained from the oxidation of methanol, inthe formation of intermediates of certain central metabolic pathwaysthat can be used, for example, in the formation of biomass. In some ofembodiments, the formaldehyde assimilation pathway comprises ahexylose-6-phosphate synthase, 6-phospho-3-hexyloisomerase,dihydroxyacetone synthase or dihydroxyacetone kinase. In certainembodiments, provided herein is a non-naturally occurring microbialorganism having a methanol metabolic pathway, wherein said organismcomprises at least one exogenous nucleic acid encoding a methanoldehydrogenase expressed in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol and/orexpressed in a sufficient amount to convert methanol to formaldehyde. Insome embodiments, the microbial organism further comprises aformaldehyde assimilation pathway. In certain embodiments, the organismfurther comprises at least one exogenous nucleic acid encoding aformaldehyde assimilation pathway enzyme expressed in a sufficientamount to produce an intermediate of glycolysis. In certain embodiments,the formaldehyde assimilation pathway enzyme is selected from the groupconsisting of a hexylose-6-phosphate synthase,6-phospho-3-hexyloisomerase, dihydroxyacetone synthase anddihydroxyacetone kinase.

In some embodiments, the organism further comprises one or more genedisruptions, occurring in one or more endogenous genes encodingprotein(s) or enzyme(s) involved in native production of ethanol,glycerol, acetate, lactate, formate, CO₂, and/or amino acids by saidmicrobial organism, wherein said one or more gene disruptions conferincreased production of succinate in said microbial organism. In someembodiments, one or more endogenous enzymes involved in nativeproduction of ethanol, glycerol, acetate, lactate, formate, CO₂ and/oramino acids by the microbial organism, has attenuated enzyme activity orexpression levels. In certain embodiments, the organism comprises fromone to twenty-five gene disruptions. In other embodiments, the organismcomprises from one to twenty gene disruptions. In some embodiments, theorganism comprises from one to fifteen gene disruptions. In otherembodiments, the organism comprises from one to ten gene disruptions. Insome embodiments, the organism comprises from one to five genedisruptions. In certain embodiments, the organism comprises 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24 or 25 gene disruptions or more.

In other aspects, provided herein are methods for producing succinate,comprising culturing any one of the non-naturally occurring microbialorganisms comprising a methanol metabolic pathway and a succinatepathway provided herein under conditions and for a sufficient period oftime to produce succinate. In certain embodiments, the organism iscultured in a substantially anaerobic culture medium.

2. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary metabolic pathways enabling the extraction ofreducing equivalents from methanol. The enzymatic transformations shownare carried out by the following enzymes: 1A) a methanolmethyltransferase, 1B) a methylenetetrahydrofolate reductase, 1C) amethylenetetrahydrofolate dehydrogenase, 1D) a methenyltetrahydrofolatecyclohydrolase, 1E) a formyltetrahydrofolate deformylase, 1F) aformyltetrahydrofolate synthetase, 1G) a formate hydrogen lyase, 1H) ahydrogenase, 1I) a formate dehydrogenase, 1J) a methanol dehydrogenase,1K) a formaldehyde activating enzyme, 1L) a formaldehyde dehydrogenase,1M) a S-(hydroxymethyl)glutathione synthase, 1N) a glutathione-dependentformaldehyde dehydrogenase, and 1O) a S-formylglutathione hydrolase. Incertain embodiments, steps K and/or M are spontaneous.

FIG. 2 shows exemplary succinate pathways, which can be used to increasesuccinate yields from carbohydrates when reducing equivalents producedby a methanol metabolic pathway provided herein are available. Forexample, pathways for the production of succinate from glucose, CO₂, andreducing equivalents (e.g., MeOH) at a theoretical yield of 2.0 molsuccinate/mol glucose are provided. The enzymatic transformations shownare carried out by the following enzymes: 2A) a phosphoenolpyruvate(PEP) carboxylase or a PEP carboxykinase; 2B) a pyruvate carboxylase;2C) a malate dehydrogenase; 2D) a malic enzyme; 2E) a fumarase; and 2F)a fumarate reductase. Succinate production can be carried out by 2A, 2C,2E and 2F; 2B, 2C, 2E and 2F; or 2D, 2E and 2F.

FIG. 3 shows an exemplary formaldehyde assimilation pathway. Theenzymatic transformations are carried out by the following enzymes: 3A)a hexylose-6-phosphate synthase, and 3B) a 6-phospho-3-hexyloisomerase.

FIG. 4 shows an exemplary formaldehyde assimilation pathway. Theenzymatic transformations are carried out by the following enzymes: 4A)a dihydroxyacetone synthase, and 4B) a dihydroxyacetone kinase.

3. DETAILED DESCRIPTION OF THE INVENTION 3.1 Definitions

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial organism's genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within a succinatebiosynthetic pathway.

As used herein, “succinate” is the ionized form of succinic acid (IUPACname butanedioic acid), and it is understood that succinate and succinicacid can be used interchangeably throughout to refer to the compound inany of its neutral or ionized forms, including any salt forms thereof.It is understood by those skilled understand that the specific form willdepend on the pH. The chemical structure of succinic acid is shownbelow:

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides, or functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” are intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

As used herein, the term “gene disruption,” or grammatical equivalentsthereof, is intended to mean a genetic alteration that renders theencoded gene product inactive or attenuated. The genetic alteration canbe, for example, deletion of the entire gene, deletion of a regulatorysequence required for transcription or translation, deletion of aportion of the gene which results in a truncated gene product, or by anyof various mutation strategies that inactivate or attenuate the encodedgene product. One particularly useful method of gene disruption iscomplete gene deletion because it reduces or eliminates the occurrenceof genetic reversions in the non-naturally occurring microorganisms ofthe invention. The phenotypic effect of a gene disruption can be a nullmutation, which can arise from many types of mutations includinginactivating point mutations, entire gene deletions, and deletions ofchromosomal segments or entire chromosomes. Specific antisense nucleicacid compounds and enzyme inhibitors, such as antibiotics, can alsoproduce null mutant phenotype, therefore being equivalent to genedisruption.

As used herein, the term “growth-coupled” when used in reference to theproduction of a biochemical product is intended to mean that thebiosynthesis of the referenced biochemical product is produced duringthe growth phase of a microorganism. In a particular embodiment, thegrowth-coupled production can be obligatory, meaning that thebiosynthesis of the referenced biochemical is an obligatory productproduced during the growth phase of a microorganism. The term“growth-coupled” when used in reference to the consumption of abiochemical is intended to mean that the referenced biochemical isconsumed during the growth phase of a microorganism.

As used herein, the term “attenuate,” or grammatical equivalentsthereof, is intended to mean to weaken, reduce or diminish the activityor amount of an enzyme or protein. Attenuation of the activity or amountof an enzyme or protein can mimic complete disruption if the attenuationcauses the activity or amount to fall below a critical level requiredfor a given pathway to function. However, the attenuation of theactivity or amount of an enzyme or protein that mimics completedisruption for one pathway, can still be sufficient for a separatepathway to continue to function. For example, attenuation of anendogenous enzyme or protein can be sufficient to mimic the completedisruption of the same enzyme or protein for production of a fattyalcohol, fatty aldehyde or fatty acid product of the invention, but theremaining activity or amount of enzyme or protein can still besufficient to maintain other pathways, such as a pathway that iscritical for the host microbial organism to survive, reproduce or grow.Attenuation of an enzyme or protein can also be weakening, reducing ordiminishing the activity or amount of the enzyme or protein in an amountthat is sufficient to increase yield of a fatty alcohol, fatty aldehydeor fatty acid product of the invention, but does not necessarily mimiccomplete disruption of the enzyme or protein.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

It is understood that when more than one nucleic acid is included in amicrobial organism that the more than one nucleic acids refers to thereferenced encoding nucleic acid or biosynthetic activity, as discussedabove. In some embodiments, the nucleic acid is an exogenous nucleicacid. In other embodiments, the nucleic acid is an endogenous nucleicacid. It is further understood, as disclosed herein, that such more thanone exogenous nucleic acids can be introduced into the host microbialorganism on separate nucleic acid molecules, on polycistronic nucleicacid molecules, or a combination thereof, and still be considered asmore than one exogenous nucleic acid. For example, as disclosed herein amicrobial organism can be engineered to express two or more exogenousnucleic acids encoding a desired pathway enzyme or protein. In the casewhere two exogenous nucleic acids encoding a desired activity areintroduced into a host microbial organism, it is understood that the twoexogenous nucleic acids can be introduced as a single nucleic acid, forexample, on a single plasmid, on separate plasmids, can be integratedinto the host chromosome at a single site or multiple sites, and stillbe considered as two exogenous nucleic acids. Similarly, it isunderstood that more than two exogenous nucleic acids can be introducedinto a host organism in any desired combination, for example, on asingle plasmid, on separate plasmids, can be integrated into the hostchromosome at a single site or multiple sites, and still be consideredas two or more exogenous nucleic acids, for example three exogenousnucleic acids. Thus, the number of referenced exogenous nucleic acids orbiosynthetic activities refers to the number of encoding nucleic acidsor the number of biosynthetic activities, not the number of separatenucleic acids introduced into the host organism.

The non-naturally occurring microbial organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having succinate biosyntheticcapability, those skilled in the art will understand with applying theteaching and guidance provided herein to a particular species that theidentification of metabolic modifications can include identification andinclusion or inactivation of orthologs. To the extent that paralogsand/or nonorthologous gene displacements are present in the referencedmicroorganism that encode an enzyme catalyzing a similar orsubstantially similar metabolic reaction, those skilled in the art alsocan utilize these evolutionally related genes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

3.2 Microbial Organisms that Utilize Reducing Equivalents Produced bythe Metabolism of Methanol

Provided herein are methanol metabolic pathways engineered to improvethe availability of reducing equivalents, which can be used for theproduction of product molecules. Exemplary product molecules include,without limitation, succinate, although given the teachings and guidanceprovided herein, it will be recognized by one skilled in the art thatany product molecule that utilizes reducing equivalents in itsproduction can exhibit enhanced production through the biosyntheticpathways provided herein.

Methanol is a relatively inexpensive organic feedstock that can bederived from synthesis gas components, CO and H₂, via catalysis.Methanol can be used as a source of reducing equivalents to increase themolar yield of product molecules from carbohydrates.

Succinate is a compound of commercial interest due to its use as aprecursor to commodity chemicals in the food, pharmaceutical, detergentand polymer industries. Biological succinate production is also a greenprocess where the greenhouse gas CO₂ must be fixed into succinate duringsugar fermentation.

Despite efforts to develop bacterial strains producing increasedsuccinate yields, many approaches previously employed have severaldrawbacks which hinder applicability in commercial settings. Forexample, many such strains generally are unstable in commercialfermentation processes due to selective pressures favoring the unalteredor wild-type parental counterparts.

Thus, there exists a need for microorganisms having commerciallybeneficial characteristics of increased production of succinate. Thepresent invention satisfies this need and provides related advantages aswell.

Accordingly, provided herein is bioderived succinate produced accordingto the methods described herein and biobased products comprising orobtained using the bioderived succinate. The biobased product cancomprise at least 5%, at least 10%, at least 20%, at least 30%, at least40% or at least 50% bioderived succinate. The biobased product cancomprises a portion of said bioderived succinate as a repeating unit.The biobased product can be a a molded product obtained by molding thebiobased product.

In numerous engineered pathways, realization of maximum product yieldsbased on carbohydrate feedstock is hampered by insufficient reducingequivalents or by loss of reducing equivalents to byproducts. Methanolis a relatively inexpensive organic feedstock that can be used togenerate reducing equivalents by employing one or more methanolmetabolic enzymes as shown in FIG. 1. The reducing equivalents producedby the metabolism of methanol by one or more of the methanol metabolicpathways can then be used to power the glucose to succinate productionpathways, for example, as shown in FIG. 2.

The product yields per C-mol of substrate of microbial cellssynthesizing reduced fermentation products such as succinate are limitedby insufficient reducing equivalents in the carbohydrate feedstock.Reducing equivalents, or electrons, can be extracted from methanol usingone or more of the enzymes described in FIG. 1. The reducing equivalentsare then passed to acceptors such as oxidized ferredoxins, oxidizedquinones, oxidized cytochromes, NAD(P)+, water, or hydrogen peroxide toform reduced ferredoxin, reduced quinones, reduced cytochromes, NAD(P)H,H₂, or water, respectively. Reduced ferredoxin, reduced quinones andNAD(P)H are particularly useful as they can serve as redox carriers forvarious Wood-Ljungdahl pathway, reductive TCA cycle, or product pathwayenzymes.

Specific examples of how additional redox availability from methanol canimprove the yield of reduced products such as succinate are shown.

Succinate can be produced solely from sugars such as glucose and CO₂with a maximum theoretical yield of 1.71 mol succinate/mol glucose or1.12 g/g.7C₆H₁₂O₆+6CO₂=12C₄H₆O₄+6H₂O

Identical or similar g/g yields are achievable on other carbohydrates(for example, xylose and arabinose).8.4C₅H₁₀O₅+6CO₂=12C₄H₆O₄+6H₂O

Alternatively, assuming that an ample source of reducing equivalents(for example, CO or H₂) is present, succinate can be produced completelyfrom CO or CO₂ via the reductive TCA (rTCA) cycle, associatedanapleurotic reactions, and enzymes for the extraction of reducingequivalents from CO and/or H₂.7CO+3H₂O═C₄H₆O₄+3CO₂7H₂+4CO₂═C₄H₆O₄+4H₂O

When both feedstocks of sugar and methanol are available, the methanolcan be utilized to generate reducing equivalents by employing one ormore of the enzymes shown in FIG. 1. The reducing equivalents generatedfrom methanol can be utilized to power the glucose to succinateproduction pathways, e.g., as shown in FIG. 2. Theoretically, allcarbons in glucose will be conserved, thus resulting in a maximaltheoretical yield to produce succinate from glucose at 2 mol succinateper mol of glucose under either aerobic or anaerobic conditions as shownin FIG. 2:C₆H₁₂O₆+0.667CH₃OH+1.333 CO₂→2C₄H₆O₄+1.333 H₂O

Supplementing carbohydrate feeds with external reducing equivalents isan attractive option for production of succinate through the reductiveTCA cycle. A reductive TCA succinate pathway is particularly useful forthe engineering of a eukaryotic organism (e.g., Saccharomycescerevisiae) for the production of succinate, as reactions associatedwith the conversion of oxaloacetate to alpha-ketoglutarate or pyruvateto acetyl-CoA are not required. Eukaryotic organisms are advantaged oversome bacterial species (e.g., Escherichia coli) in that they cantolerate lower pH conditions. Production of succinate at a pH lower thanthe pKa's of the acid groups (pK_(a1)=4.2, pK_(a2)=5.6) is desirablesince a higher percentage of final product will be in the acid form(i.e., succinic acid) and not the salt form.

A major challenge associated with engineering a eukaryotic organism toachieve a high yield of succinate from carbohydrates alone is thatseveral requisite enzymes are not exclusively localized to the cytosol.In fact, enzymes such as pyruvate dehydrogenase and citrate synthase arepredominantly mitochondrial or peroxisomal. Thus, engineering aeukaryotic organism to achieve a high yield of succinate would requireshuttling of metabolic precursors across cellular compartments orextensive strain engineering to localize several of the requisiteenzymes to one compartment, preferably the cytosol. This embodimentprovides a means of simplifying the pathway engineering by limiting thenumber of TCA cycle enzymes that are required to carry high flux. Onecan thus envision the development of a succinate producing microorganismin which most, if not all, of the enzymes in FIG. 1 are cytosolic.

In a first aspect, provided herein is a non-naturally occurringmicrobial organism having a methanol metabolic pathway, wherein saidorganism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme expressed in a sufficient amount toenhance the availability of reducing equivalents in the presence ofmethanol. In certain embodiments, the methanol metabolic pathwaycomprises one or more enzymes selected from the group consisting of amethanol methyltransferase; a methylenetetrahydrofolate reductase; amethylenetetrahydrofolate dehydrogenase; a methenyltetrahydrofolatecyclohydrolase; a formyltetrahydrofolate deformylase; aformyltetrahydrofolate synthetase; a formate hydrogen lyase; ahydrogenase; a formate dehydrogenase; a methanol dehydrogenase; aformaldehyde activating enzyme; a formaldehyde dehydrogenase; aS-(hydroxymethyl)glutathione synthase; a glutathione-dependentformaldehyde dehydrogenase; and an S-formylglutathione hydrolase. Suchorganisms advantageously allow for the production of reducingequivalents, which can then be used by the organism for the productionof succinate using any one of the succinate pathways provided herein.

In certain embodiments, the methanol metabolic pathway comprises 1A, 1B,1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or any combinationof 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, and 1O,thereof, wherein 1A is a methanol methyltransferase; 1B is amethylenetetrahydrofolate reductase; 1C is a methylenetetrahydrofolatedehydrogenase; 1D is a methenyltetrahydrofolate cyclohydrolase; 1E is aformyltetrahydrofolate deformylase; 1F is a formyltetrahydrofolatesynthetase; 1G is a formate hydrogen lyase; 1H is a hydrogenase, 1I is aformate dehydrogenase; 1J is a methanol dehydrogenase; 1K is aformaldehyde activating enzyme; 1L is a formaldehyde dehydrogenase; 1Mis a S-(hydroxymethyl)glutathione synthase; 1N is glutathione-dependentformaldehyde dehydrogenase; and 1O is S-formylglutathione hydrolase. Insome embodiments, 1K is spontaneous. In other embodiments, 1K is aformaldehyde activating enzyme. In some embodiments, 1M is spontaneous.In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase.

In one embodiment, the methanol metabolic pathway comprises 1A. Inanother embodiment, the methanol metabolic pathway comprises 1B. Inanother embodiment, the methanol metabolic pathway comprises 1C. In yetanother embodiment, the methanol metabolic pathway comprises 1D. In oneembodiment, the methanol metabolic pathway comprises 1E. In anotherembodiment, the methanol metabolic pathway comprises 1F. In anotherembodiment, the methanol metabolic pathway comprises 1G. In yet anotherembodiment, the methanol metabolic pathway comprises 1H. In oneembodiment, the methanol metabolic pathway comprises 1I. In anotherembodiment, the methanol metabolic pathway comprises 1J. In anotherembodiment, the methanol metabolic pathway comprises 1K. In yet anotherembodiment, the methanol metabolic pathway comprises 1L. In yet anotherembodiment, the methanol metabolic pathway comprises 1M. In anotherembodiment, the methanol metabolic pathway comprises 1N. In yet anotherembodiment, the methanol metabolic pathway comprises 1O. Any combinationof two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen or fifteen methanol metabolic pathway enzymes 1A, 1B,1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, and 1O is alsocontemplated.

In some embodiments, the methanol metabolic pathway is a methanolmetabolic pathway depicted in FIG. 1.

In one aspect, provided herein is a non-naturally occurring microbialorganism having a methanol metabolic pathway, wherein said organismcomprises at least one exogenous nucleic acid encoding a methanolmetabolic pathway enzyme expressed in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol,wherein said methanol metabolic pathway comprises: (i) 1A and 1B, (ii)1J; or (iii) 1J and 1K. In one embodiment, the methanol metabolicpathway comprises 1A and 1B. In another embodiment, the methanolmetabolic pathway comprises 1J. In one embodiment, the methanolmetabolic pathway comprises 1J and 1K. In certain embodiments, themethanol metabolic pathway comprises 1A, 1B, 1C, 1D, and 1E. In someembodiments. the methanol metabolic pathway comprises 1A, 1B, 1C, 1D and1F. In some embodiments, the methanol metabolic pathway comprises 1J,1C, 1D and 1E. In one embodiment, the methanol metabolic pathwaycomprises 1J, 1C, 1D and 1F. In another embodiment, the methanolmetabolic pathway comprises 1J and 1L. In yet another embodiment, themethanol metabolic pathway comprises 1J, 1M, 1N and 1O. In certainembodiments, the methanol metabolic pathway comprises 1J, 1N and 1O. Insome embodiments, the methanol metabolic pathway comprises 1J, 1K, 1C,1D and 1E. In one embodiment, the methanol metabolic pathway comprises1J, 1K, 1C, 1D and 1F. In some embodiments, 1K is spontaneous. In otherembodiments, 1K is a formaldehyde activating enzyme. In someembodiments, 1M is spontaneous. In other embodiments, 1M is aS-(hydroxymethyl)glutathione synthase.

In certain embodiments, the methanol metabolic pathway comprises 1I. Incertain embodiments, the methanol metabolic pathway comprises 1A, 1B,1C, 1D, 1E and 1I. In some embodiments. the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F and 1I. In some embodiments, the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E and 1I. In one embodiment,the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1I. Inanother embodiment, the methanol metabolic pathway comprises 1J, 1L and1I. In yet another embodiment, the methanol metabolic pathway comprises1J, 1M, 1N, 1O and 1I. In certain embodiments, the methanol metabolicpathway comprises 1J, 1N, 1O and 1I. In some embodiments, the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I. In oneembodiment, the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1Fand 1I. In some embodiments, 1K is spontaneous. In other embodiments, 1Kis a formaldehyde activating enzyme. In some embodiments, 1M isspontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathionesynthase.

In certain embodiments, the methanol metabolic pathway comprises 1G. Incertain embodiments, the methanol metabolic pathway comprises 1A, 1B,1C, 1D, 1E and 1G. In some embodiments. the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F and 1G. In some embodiments, the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E and 1G. In one embodiment,the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G. Inanother embodiment, the methanol metabolic pathway comprises 1 J, 1L and1G. In yet another embodiment, the methanol metabolic pathway comprises1J, 1M, 1N, 1O and 1G. In certain embodiments, the methanol metabolicpathway comprises 1J, 1N, 1O and 1G. In some embodiments, the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G. In oneembodiment, the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1Fand 1G. In some embodiments, 1K is spontaneous. In other embodiments, 1Kis a formaldehyde activating enzyme. In some embodiments, 1M isspontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathionesynthase.

In certain embodiments, the methanol metabolic pathway comprises 1G and1H. In certain embodiments, the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1E, 1G and 1H. In some embodiments. the methanol metabolicpathway comprises 1A, 1B, 1C, 1D, 1F, 1G and 1H. In some embodiments,the methanol metabolic pathway comprises 1J, 1C, 1D, 1E, 1G and 1H. Inone embodiment, the methanol metabolic pathway comprises 1J, 1C, 1D, 1F,1G and 1H. In another embodiment, the methanol metabolic pathwaycomprises 1J, 1L, 1G and 1H. In yet another embodiment, the methanolmetabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H. In certainembodiments, the methanol metabolic pathway comprises 1J, 1N, 1O, 1G and1H. In some embodiments, the methanol metabolic pathway comprises 1J,1K, 1C, 1D, 1E, 1G and 1H. In one embodiment, the methanol metabolicpathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H. In some embodiments, 1Kis spontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1Mis a S-(hydroxymethyl)glutathione synthase.

In certain embodiments, the formation of 5-hydroxymethylglutathione fromformaldehyde is spontaneous (see, e.g., FIG. 1, step M). In someembodiments, the formation of 5-hydroxymethylglutathione fromformaldehyde is catalyzed by a S-(hydroxymethyl)glutathione synthase(see, e.g., FIG. 1, step M). In certain embodiments, the formation ofmethylene-THF from formaldehyde is spontaneous (see, e.g., FIG. 1, stepK). In certain embodiments, the formation of methylene-THF fromformaldehyde is catalyzed by a formaldehyde activating enzyme (see,e.g., FIG. 1, step K).

In certain embodiments, the organism comprises two, three, four, five,six or seven exogenous nucleic acids, each encoding a methanol metabolicpathway enzyme. In certain embodiments, the organism comprises twoexogenous nucleic acids, each encoding a methanol metabolic pathwayenzyme. In certain embodiments, the organism comprises three exogenousnucleic acids, each encoding a methanol metabolic pathway enzyme. Incertain embodiments, the organism comprises four exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism comprises five exogenous nucleic acids, eachencoding a methanol metabolic pathway enzyme. In certain embodiments,the organism comprises six exogenous nucleic acids, each encoding amethanol metabolic pathway enzyme. In certain embodiments, the organismcomprises seven exogenous nucleic acids, each encoding a methanolmetabolic pathway enzyme.

Any non-naturally occurring eukaryotic organism comprising a methanolmetabolic pathway and engineered to comprise a methanol metabolicpathway enzyme, such as those provided herein, can be engineered tofurther comprise one or more succinate pathway enzymes.

In one embodiment, the non-naturally occurring microbial organismfurther comprises a succinate pathway. In some embodiments, the organismfurther comprises at least one nucleic acid encoding a succinate pathwayenzyme expressed in a sufficient amount to produce succinate. In someembodiments, the nucleic acid is an exogenous nucleic acid. In otherembodiments, the nucleic acid is an endogenous nucleic acid. In certainembodiments, the succinate pathway enzyme is selected from the groupconsisting of a PEP carboxylase or a PEP carboxykinase; a pyruvatecarboxylase; a malate dehydrogenase; a malic enzyme; a fumarase; and afumarate reductase.

In some embodiments, the non-naturally occurring microbial organismshaving a succinate pathway includes a set of succinate pathway enzymes.

Enzymes, genes and methods for engineering pathways from glucose tovarious products, such as succinate, into a microorganism, are now knownin the art (see, e.g., U.S. Publ. No. 2011/0201089, which is hereinincorporated by reference in its entirety). A set of succinate pathwayenzymes represents a group of enzymes that can convert pyruvate orphosphoenolpyruvate to succinate, for example, as shown in FIG. 2. Theadditional reducing equivalents obtained from the methanol metabolicpathways, as disclosed herein, improve the yields of all these productswhen utilizing carbohydrate-based feedstock.

Exemplary enzymes for the conversion glucose to succinate (e.g., viapyruvate) include a phosphoenolpyruvate (PEP) carboxylase or a PEPcarboxykinase (FIG. 2, step A); a pyruvate carboxylase (FIG. 2, step B);a malate dehydrogenase (FIG. 2, step C); a malic enzyme (FIG. 2, stepD); a fumarase (FIG. 2, step E); and a fumarate reductase (FIG. 2, stepF).

In one aspect, provided herein is a non-naturally occurring microbialorganism, comprising (1) a methanol metabolic pathway, wherein saidorganism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol; and(2) a succinate pathway. In some embodiments, the organism furthercomprises at least one nucleic acid (e.g., an endogenous and/orexogenous nucleic acid) encoding a succinate pathway enzyme expressed ina sufficient amount to produce succinate. In one embodiment, the atleast one exogenous nucleic acid encoding the methanol metabolic pathwayenzyme enhances the availability of reducing equivalents in the presenceof methanol in a sufficient amount to increase the amount of succinateproduced by the non-naturally microbial organism. In some embodiments,the methanol metabolic pathway comprises any of the various combinationsof methanol metabolic pathway enzymes described above or elsewhereherein.

In certain embodiments, (1) the methanol metabolic pathway comprises:1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or anycombination of 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N,or 1O, thereof, wherein 1A is a methanol methyltransferase; 1B is amethylenetetrahydrofolate reductase; 1C is a methylenetetrahydrofolatedehydrogenase; 1D is a methenyltetrahydrofolate cyclohydrolase; 1E is aformyltetrahydrofolate deformylase; 1F is a formyltetrahydrofolatesynthetase; 1G is a formate hydrogen lyase; 1H is a hydrogenase, 1I is aformate dehydrogenase; 1J is a methanol dehydrogenase; 1K is spontaneousor formaldehyde activating enzyme; 1L is a formaldehyde dehydrogenase;1M is spontaneous or a S-(hydroxymethyl)glutathione synthase; 1N isglutathione-dependent formaldehyde dehydrogenase and 1O isS-formylglutathione hydrolase; and (2) the succinate pathway comprises2A, 2B, 2C, 2D, 2E or 2F, or any combination thereof, wherein 2A is aPEP carboxylase or a PEP carboxykinase; 2B is a pyruvate carboxylase; 2Cis a malate dehydrogenase; 2D is a malic enzyme; 2E is a fumarase; and2F is a fumarate reductase. In some embodiments, 1K is spontaneous. Inother embodiments, 1K is a formaldehyde activating enzyme. In someembodiments, 1M is spontaneous. In other embodiments, 1M is aS-(hydroxymethyl)glutathione synthase. In some embodiments, 2A is a PEPcarboxylase. In other embodiments, 2A is a PEP carboxykinase.

In one embodiment, the succinate pathway comprises 2A. In anotherembodiment, the succinate pathway comprises 2B. In an embodiment, thesuccinate pathway comprises 2C. In another embodiment, the succinatepathway comprises 2D. In another embodiment, the succinate pathwaycomprises 2E. In an embodiment, the succinate pathway comprises 2F. Anycombination of two, three, four, five or six succinate pathway enzymes2A, 2B, 2C, 2D, 2E and 2F is also contemplated. In some embodiments, 2Ais a PEP carboxylase. In other embodiments, 2A is a PEP carboxykinase.

In some embodiments, the methanol metabolic pathway is a methanolmetabolic pathway depicted in FIG. 1, and the succinate pathway is asuccinate pathway depicted in FIG. 2.

Exemplary sets of succinate pathway enzymes to convert glucose tosuccinate (e.g., via pyruvate or phosphoenolpyruvate) according to FIG.2 include (i) 2A, 2C, 2E and 2F; (ii) 2B, 2C, 2E and 2F; and (iii) 2D,2E and 2F. In some embodiments, 2A is a PEP carboxylase. In otherembodiments, 2A is a PEP carboxykinase.

In one embodiment, (1) the methanol metabolic pathway comprises 1A and1B; and (2) the succinate pathway comprises 2A, 2C, 2E and 2F. Inanother embodiment, (1) the methanol metabolic pathway comprises 1J; and(2) the succinate pathway comprises 2A, 2C, 2E and 2F. In oneembodiment, (1) the methanol metabolic pathway comprises 1J and 1K; and(2) the succinate pathway comprises 2A, 2C, 2E and 2F. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, and 1E; and (2) the succinate pathway comprises 2A, 2C, 2E and 2F.In some embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D and 1F; and (2) the succinate pathway comprises 2A, 2C, 2Eand 2F. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D and 1E; and (2) the succinate pathway comprises 2A,2C, 2E and 2F. In one embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D and 1F; and (2) the succinate pathway comprises 2A,2C, 2E and 2F. In another embodiment, (1) the methanol metabolic pathwaycomprises 1J and 1L; and (2) the succinate pathway comprises 2A, 2C, 2Eand 2F. In yet another embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1M, 1N and 1O; and (2) the succinate pathway comprises 2A,2C, 2E and 2F. In certain embodiments, (1) the methanol metabolicpathway comprises 1J, 1N and 1O; and (2) the succinate pathway comprises2A, 2C, 2E and 2F. In some embodiments, (1) the methanol metabolicpathway comprises 1J, 1K, 1C, 1D and 1E; and (2) the succinate pathwaycomprises 2A, 2C, 2E and 2F. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D and 1F; and (2) the succinatepathway comprises 2A, 2C, 2E and 2F. In certain embodiments, (1) themethanol metabolic pathway comprises 1I; and (2) the succinate pathwaycomprises 2A, 2C, 2E and 2F. In certain embodiments, (1) the methanolmetabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1I; and (2) thesuccinate pathway comprises 2A, 2C, 2E and 2F. In some embodiments, (1)the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1I; and(2) the succinate pathway comprises 2A, 2C, 2E and 2F. In someembodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1Eand 1I; and (2) the succinate pathway comprises 2A, 2C, 2E and 2F. Inone embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D,1F and 1I; and (2) the succinate pathway comprises 2A, 2C, 2E and 2F. Inanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1Land 1I; and (2) the succinate pathway comprises 2A, 2C, 2E and 2F. Inyet another embodiment, (1) the methanol metabolic pathway comprises 1J,1M, 1N, 1O and 1I; and (2) the succinate pathway comprises 2A, 2C, 2Eand 2F. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1N, 1O and 1I; and (2) the succinate pathway comprises 2A,2C, 2E and 2F. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D, 1E and 1I; and (2) the succinate pathwaycomprises 2A, 2C, 2E and 2F. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1I; and (2) thesuccinate pathway comprises 2A, 2C, 2E and 2F. In certain embodiments,(1) the methanol metabolic pathway comprises 1G; and (2) the succinatepathway comprises 2A, 2C, 2E and 2F. In certain embodiments, (1) themethanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1G; and (2)the succinate pathway comprises 2A, 2C, 2E and 2F. In some embodiments,(1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1G;and (2) the succinate pathway comprises 2A, 2C, 2E and 2F. In someembodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1Eand 1G; and (2) the succinate pathway comprises 2A, 2C, 2E and 2F. Inone embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D,1F and 1G; and (2) the succinate pathway comprises 2A, 2C, 2E and 2F. Inanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1Land 1G; and (2) the succinate pathway comprises 2A, 2C, 2E and 2F. Inyet another embodiment, (1) the methanol metabolic pathway comprises 1J,1M, 1N, 1O and 1G; and (2) the succinate pathway comprises 2A, 2C, 2Eand 2F. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1N, 1O and 1G; and (2) the succinate pathway comprises 2A,2C, 2E and 2F. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the succinate pathwaycomprises 2A, 2C, 2E and 2F. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G; and (2) thesuccinate pathway comprises 2A, 2C, 2E and 2F. In certain embodiments,(1) the methanol metabolic pathway comprises 1G and 1H; and (2) thesuccinate pathway comprises 2A, 2C, 2E and 2F. In certain embodiments,(1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E, 1G and1H; and (2) the succinate pathway comprises 2A, 2C, 2E and 2F. In someembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, 1F, 1G and 1H; and (2) the succinate pathway comprises 2A, 2C, 2Eand 2F. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the succinate pathwaycomprises 2A, 2C, 2E and 2F. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2) thesuccinate pathway comprises 2A, 2C, 2E and 2F. In another embodiment,(1) the methanol metabolic pathway comprises 1J, 1L, 1G and 1H; and (2)the succinate pathway comprises 2A, 2C, 2E and 2F. In yet anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O,1G and 1H; and (2) the succinate pathway comprises 2A, 2C, 2E and 2F. Incertain embodiments, (1) the methanol metabolic pathway comprises 1J,1N, 1O, 1G and 1H; and (2) the succinate pathway comprises 2A, 2C, 2Eand 2F. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and (2) the succinate pathwaycomprises 2A, 2C, 2E and 2F. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) thesuccinate pathway comprises 2A, 2C, 2E and 2F. In some embodiments, 1Kis spontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1Mis a S-(hydroxymethyl)glutathione synthase. In some embodiments, 2A is aPEP carboxylase. In other embodiments, 2A is a PEP carboxykinase.

In one embodiment, (1) the methanol metabolic pathway comprises 1A and1B; and (2) the succinate pathway comprises 2B, 2C, 2E and 2F. Inanother embodiment, (1) the methanol metabolic pathway comprises 1J; and(2) the succinate pathway comprises 2B, 2C, 2E and 2F. In oneembodiment, (1) the methanol metabolic pathway comprises 1J and 1K; and(2) the succinate pathway comprises 2B, 2C, 2E and 2F. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, and 1E; and (2) the succinate pathway comprises 2B, 2C, 2E and 2F.In some embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D and 1F; and (2) the succinate pathway comprises 2B, 2C, 2Eand 2F. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D and 1E; and (2) the succinate pathway comprises 2B,2C, 2E and 2F. In one embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D and 1F; and (2) the succinate pathway comprises 2B,2C, 2E and 2F. In another embodiment, (1) the methanol metabolic pathwaycomprises 1J and 1L; and (2) the succinate pathway comprises 2B, 2C, 2Eand 2F. In yet another embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1M, 1N and 1O; and (2) the succinate pathway comprises 2B,2C, 2E and 2F. In certain embodiments, (1) the methanol metabolicpathway comprises 1J, 1N and 1O; and (2) the succinate pathway comprises2B, 2C, 2E and 2F. In some embodiments, (1) the methanol metabolicpathway comprises 1J, 1K, 1C, 1D and 1E; and (2) the succinate pathwaycomprises 2B, 2C, 2E and 2F. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D and 1F; and (2) the succinatepathway comprises 2B, 2C, 2E and 2F. In certain embodiments, (1) themethanol metabolic pathway comprises 1I; and (2) the succinate pathwaycomprises 2B, 2C, 2E and 2F. In certain embodiments, (1) the methanolmetabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1I; and (2) thesuccinate pathway comprises 2B, 2C, 2E and 2F. In some embodiments, (1)the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1I; and(2) the succinate pathway comprises 2B, 2C, 2E and 2F. In someembodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1Eand 1I; and (2) the succinate pathway comprises 2B, 2C, 2E and 2F. Inone embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D,1F and 1I; and (2) the succinate pathway comprises 2B, 2C, 2E and 2F. Inanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1Land 1I; and (2) the succinate pathway comprises 2B, 2C, 2E and 2F. Inyet another embodiment, (1) the methanol metabolic pathway comprises 1J,1M, 1N, 1O and 1I; and (2) the succinate pathway comprises 2B, 2C, 2Eand 2F. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1N, 1O and 1I; and (2) the succinate pathway comprises 2B,2C, 2E and 2F. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D, 1E and 1I; and (2) the succinate pathwaycomprises 2B, 2C, 2E and 2F. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1I; and (2) thesuccinate pathway comprises 2B, 2C, 2E and 2F. In certain embodiments,(1) the methanol metabolic pathway comprises 1G; and (2) the succinatepathway comprises 2B, 2C, 2E and 2F. In certain embodiments, (1) themethanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1G; and (2)the succinate pathway comprises 2B, 2C, 2E and 2F. In some embodiments,(1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1G;and (2) the succinate pathway comprises 2B, 2C, 2E and 2F. In someembodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1Eand 1G; and (2) the succinate pathway comprises 2B, 2C, 2E and 2F. Inone embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D,1F and 1G; and (2) the succinate pathway comprises 2B, 2C, 2E and 2F. Inanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1Land 1G; and (2) the succinate pathway comprises 2B, 2C, 2E and 2F. Inyet another embodiment, (1) the methanol metabolic pathway comprises 1J,1M, 1N, 1O and 1G; and (2) the succinate pathway comprises 2B, 2C, 2Eand 2F. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1N, 1O and 1G; and (2) the succinate pathway comprises 2B,2C, 2E and 2F. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the succinate pathwaycomprises 2B, 2C, 2E and 2F. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G; and (2) thesuccinate pathway comprises 2B, 2C, 2E and 2F. In certain embodiments,(1) the methanol metabolic pathway comprises 1G and 1H; and (2) thesuccinate pathway comprises 2B, 2C, 2E and 2F. In certain embodiments,(1) the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E, 1G and1H; and (2) the succinate pathway comprises 2B, 2C, 2E and 2F. In someembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, 1F, 1G and 1H; and (2) the succinate pathway comprises 2B, 2C, 2Eand 2F. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the succinate pathwaycomprises 2B, 2C, 2E and 2F. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2) thesuccinate pathway comprises 2B, 2C, 2E and 2F. In another embodiment,(1) the methanol metabolic pathway comprises 1J, 1L, 1G and 1H; and (2)the succinate pathway comprises 2B, 2C, 2E and 2F. In yet anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O,1G and 1H; and (2) the succinate pathway comprises 2B, 2C, 2E and 2F. Incertain embodiments, (1) the methanol metabolic pathway comprises 1J,1N, 1O, 1G and 1H; and (2) the succinate pathway comprises 2B, 2C, 2Eand 2F. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and (2) the succinate pathwaycomprises 2B, 2C, 2E and 2F. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) thesuccinate pathway comprises 2B, 2C, 2E and 2F. In some embodiments, 1Kis spontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1Mis a S-(hydroxymethyl)glutathione synthase.

In one embodiment, (1) the methanol metabolic pathway comprises 1A and1B; and (2) the succinate pathway comprises 2D, 2E and 2F. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J; and (2) thesuccinate pathway comprises 2D, 2E and 2F. In one embodiment, (1) themethanol metabolic pathway comprises 1J and 1K; and (2) the succinatepathway comprises 2D, 2E and 2F. In certain embodiments, (1) themethanol metabolic pathway comprises 1A, 1B, 1C, 1D, and 1E; and (2) thesuccinate pathway comprises 2D, 2E and 2F. In some embodiments, (1) themethanol metabolic pathway comprises 1A, 1B, 1C, 1D and 1F; and (2) thesuccinate pathway comprises 2D, 2E and 2F. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1C, 1D and 1E; and (2) thesuccinate pathway comprises 2D, 2E and 2F. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1C, 1D and 1F; and (2) thesuccinate pathway comprises 2D, 2E and 2F. In another embodiment, (1)the methanol metabolic pathway comprises 1J and 1L; and (2) thesuccinate pathway comprises 2D, 2E and 2F. In yet another embodiment,(1) the methanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2)the succinate pathway comprises 2D, 2E and 2F. In certain embodiments,(1) the methanol metabolic pathway comprises 1J, 1N and 1O; and (2) thesuccinate pathway comprises 2D, 2E and 2F. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1E; and (2) thesuccinate pathway comprises 2D, 2E and 2F. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1F; and (2) thesuccinate pathway comprises 2D, 2E and 2F. In certain embodiments, (1)the methanol metabolic pathway comprises 1I; and (2) the succinatepathway comprises 2D, 2E and 2F. In certain embodiments, (1) themethanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1I; and (2)the succinate pathway comprises 2D, 2E and 2F. In some embodiments, (1)the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1I; and(2) the succinate pathway comprises 2D, 2E and 2F. In some embodiments,(1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and(2) the succinate pathway comprises 2D, 2E and 2F. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1I; and(2) the succinate pathway comprises 2D, 2E and 2F. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1I;and (2) the succinate pathway comprises 2D, 2E and 2F. In yet anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1Oand 1I; and (2) the succinate pathway comprises 2D, 2E and 2F. Incertain embodiments, (1) the methanol metabolic pathway comprises 1J,1N, 1O and 1I; and (2) the succinate pathway comprises 2D, 2E and 2F. Insome embodiments, (1) the methanol metabolic pathway comprises 1J, 1K,1C, 1D, 1E and 1I; and (2) the succinate pathway comprises 2D, 2E and2F. In one embodiment, (1) the methanol metabolic pathway comprises 1J,1K, 1C, 1D, 1F and 1I; and (2) the succinate pathway comprises 2D, 2Eand 2F. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1G; and (2) the succinate pathway comprises 2D, 2E and 2F. Incertain embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1E and 1G; and (2) the succinate pathway comprises 2D, 2Eand 2F. In some embodiments, (1) the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F and 1G; and (2) the succinate pathwaycomprises 2D, 2E and 2F. In some embodiments, (1) the methanol metabolicpathway comprises 1J, 1C, 1D, 1E and 1G; and (2) the succinate pathwaycomprises 2D, 2E and 2F. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1C, 1D, 1F and 1G; and (2) the succinate pathwaycomprises 2D, 2E and 2F. In another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1L and 1G; and (2) the succinate pathwaycomprises 2D, 2E and 2F. In yet another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1M, 1N, 1O and 1G; and (2) the succinatepathway comprises 2D, 2E and 2F. In certain embodiments, (1) themethanol metabolic pathway comprises 1J, 1N, 1O and 1G; and (2) thesuccinate pathway comprises 2D, 2E and 2F. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2)the succinate pathway comprises 2D, 2E and 2F. In one embodiment, (1)the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G; and(2) the succinate pathway comprises 2D, 2E and 2F. In certainembodiments, (1) the methanol metabolic pathway comprises 1G and 1H; and(2) the succinate pathway comprises 2D, 2E and 2F. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, 1E, 1G and 1H; and (2) the succinate pathway comprises 2D, 2E and2F. In some embodiments, (1) the methanol metabolic pathway comprises1A, 1B, 1C, 1D, 1F, 1G and 1H; and (2) the succinate pathway comprises2D, 2E and 2F. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the succinate pathwaycomprises 2D, 2E and 2F. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2) the succinatepathway comprises 2D, 2E and 2F. In another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1L, 1G and 1H; and (2) the succinatepathway comprises 2D, 2E and 2F. In yet another embodiment, (1) themethanol metabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H; and (2)the succinate pathway comprises 2D, 2E and 2F. In certain embodiments,(1) the methanol metabolic pathway comprises 1J, 1N, 1O, 1G and 1H; and(2) the succinate pathway comprises 2D, 2E and 2F. In some embodiments,(1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and1H; and (2) the succinate pathway comprises 2D, 2E and 2F. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F, 1G and 1H; and (2) the succinate pathway comprises 2D, 2E and 2F. Insome embodiments, 1K is spontaneous. In other embodiments, 1K is aformaldehyde activating enzyme. In some embodiments, 1M is spontaneous.In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase.

In one embodiment, the non-naturally occurring microbial organismcomprises (1) a methanol metabolic pathway comprising 1A and 1B; 1J; 1Jand 1K; 1A, 1B, 1C, 1D, and 1E; 1A, 1B, 1C, 1D and 1F; 1J, 1C, 1D and1E; 1J, 1C, 1D and 1F; 1J and 1L; 1J, 1M, 1N and 1O; 1J, 1N and 1O; 1J,1K, 1C, 1D and 1E; 1J, 1K, 1C, 1D and 1F; 1I; 1A, 1B, 1C, 1D, 1E and 1I;1A, 1B, 1C, 1D, 1F and 1I; 1J, 1C, 1D, 1E and 1I; 1J, 1C, 1D, 1F and 1I;1J, 1L and 1I; 1J, 1M, 1N, 1O and 1I; 1J, 1N, 1O and 1I; 1J, 1K, 1C, 1D,1E and 1I; 1J, 1K, 1C, 1D, 1F and 1I; 1G; 1A, 1B, 1C, 1D, 1E and 1G; 1A,1B, 1C, 1D, 1F and 1G; 1J, 1C, 1D, 1E and 1G; 1J, 1C, 1D, 1F and 1G; 1J,1L and 1G; 1J, 1M, 1N, 1O and 1G; 1J, 1N, 1O and 1G; 1J, 1K, 1C, 1D, 1Eand 1G; 1J, 1K, 1C, 1D, 1F and 1G; 1G and 1H; 1A, 1B, 1C, 1D, 1E, 1G and1H; 1A, 1B, 1C, 1D, 1F, 1G and 1H; 1J, 1C, 1D, 1E, 1G and 1H; 1J, 1C,1D, 1F, 1G and 1H; 1J, 1L, 1G and 1H; 1J, 1M, 1N, 1O, 1G and 1H; 1J, 1N,1O, 1G and 1H; 1J, 1K, 1C, 1D, 1E, 1G and 1H; or 1J, 1K, 1C, 1D, 1F, 1Gand 1H; and (2) a succinate pathway. In some embodiments, 1K isspontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1Mis a S-(hydroxymethyl)glutathione synthase.

Any methanol metabolic pathway provided herein can be combined with anysuccinate pathway provided herein.

Also provided herein are exemplary pathways, which utilize formaldehydeproduced from the oxidation of methanol (e.g., as provided in FIG. 1,step J) in the formation of intermediates of certain central metabolicpathways that can be used for the formation of biomass. One exemplaryformaldehyde assimilation pathway that can utilize formaldehyde producedfrom the oxidation of methanol (e.g., as provided in FIG. 1) is shown inFIG. 3, which involves condensation of formaldehyde andD-ribulose-5-phosphate to form hexylose-6-phosphate (h6p) byhexylose-6-phosphate synthase (FIG. 3, step A). The enzyme can use Mg²⁺or Mn²⁺ for maximal activity, although other metal ions are useful, andeven non-metal-ion-dependent mechanisms are contemplated. H6p isconverted into fructose-6-phosphate by 6-phospho-3-hexyloisomerase (FIG.3, step B). Another exemplary pathway that involves the detoxificationand assimilation of formaldehyde produced from the oxidation of methanol(e.g., as provided in FIG. 1) is shown in FIG. 4 and proceeds throughdihydroxyacetone. Dihydroxyacetone synthase is a special transketolasethat first transfers a glycoaldehyde group from xylulose-5-phosphate toformaldehyde, resulting in the formation of dihydroxyacetone (DHA) andglyceraldehyde-3-phosphate (G3P), which is an intermediate in glycolysis(FIG. 4, step A). The DHA obtained from DHA synthase is then furtherphosphorylated to form DHA phosphate by a DHA kinase (FIG. 4, step B).DHAP can be assimilated into glycolysis and several other pathways.Rather than converting formaldehyde to formate and on to CO₂ off-gassed,the pathways provided in FIGS. 3 and 4 show that carbon is assimilated,going into the final product.

Thus, in one embodiment, an organism having a methanol metabolicpathway, either alone or in combination with a succinate pathway, asprovided herein, further comprises a formaldehyde assimilation pathwaythat utilizes formaldehyde, e.g., obtained from the oxidation ofmethanol, in the formation of intermediates of certain central metabolicpathways that can be used, for example, in the formation of biomass. Insome of embodiments, the formaldehyde assimilation pathway comprises 3Aor 3B, wherein 3A is a hexylose-6-phosphate synthase and 3B is a6-phospho-3-hexyloisomerase In other embodiments, the formaldehydeassimilation pathway comprises 4A or 4B, wherein 4A is adihydroxyacetone synthase and 4B is a dihydroxyacetone kinase.

In certain embodiments, provided herein is a non-naturally occurringmicrobial organism having a methanol metabolic pathway, wherein saidorganism comprises at least one exogenous nucleic acid encoding amethanol dehydrogenase (1J) expressed in a sufficient amount to enhancethe availability of reducing equivalents in the presence of methanoland/or expressed in a sufficient amount to convert methanol toformaldehyde. In some embodiments, the microbial organism furthercomprises a formaldehyde assimilation pathway. In certain embodiments,the organism further comprises at least one exogenous nucleic acidencoding a formaldehyde assimilation pathway enzyme expressed in asufficient amount to produce an intermediate of glycolysis and/or ametabolic pathway that can be used, for example, in the formation ofbiomass. In certain embodiments, the formaldehyde assimilation pathwayenzyme is selected from the group consisting of a hexylose-6-phosphatesynthase (3A), 6-phospho-3-hexyloisomerase (3B), dihydroxyacetonesynthase (4A) and dihydroxyacetone kinase (4B).

In one aspect, provided herein is a non-naturally occurring microbialorganism, comprising (1) a methanol metabolic pathway, wherein saidorganism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol and/orexpressed in a sufficient amount to convert methanol to formaldehyde;and (2) a formaldehyde assimilation pathway, wherein said organismcomprises at least one exogenous nucleic acid encoding a formaldehydeassimilation pathway enzyme expressed in a sufficient amount to producean intermediate of glycolysis and/or a metabolic pathway that can beused, for example, in the formation of biomass. In specific embodiments,the methanol metabolic pathway comprises a methanol dehydrogenase (1J).In certain embodiments, the formaldehyde assimilation pathway enzyme is3A, and the intermediate is a hexylose-6-phosphate, afructose-6-phosphate, or a combination thereof. In other embodiments,the formaldehyde assimilation pathway enzyme is 3B, and the intermediateis a hexylose-6-phosphate, a fructose-6-phosphate, or a combinationthereof. In yet other embodiments, the formaldehyde assimilation pathwayenzyme is 3A and 3B, and the intermediate is a hexylose-6-phosphate, afructose-6-phosphate, or a combination thereof. In some embodiments, theformaldehyde assimilation pathway enzyme is 4A, and the intermediate isa dihydroxyacetone (DHA), a dihydroxyacetone phosphate, or a combinationthereof. In other embodiments, the formaldehyde assimilation pathwayenzyme is 4B, and the intermediate is a DHA, a dihydroxyacetonephosphate, or a combination thereof. In yet other embodiments, theformaldehyde assimilation pathway enzyme is 4A and 4B, and theintermediate is a DHA, a dihydroxyacetone phosphate, or a combinationthereof. In one embodiment, the at least one exogenous nucleic acidencoding the methanol metabolic pathway enzyme, in the presence ofmethanol, sufficiently enhances the availability of reducing equivalentsand sufficiently increases formaldehyde assimilation to increase theproduction of succinate or other products described herein by thenon-naturally microbial organism. In some embodiments, the methanolmetabolic pathway comprises any of the various combinations of methanolmetabolic pathway enzymes described above or elsewhere herein.

In certain embodiments, (1) the methanol metabolic pathway comprises:1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or anycombination of 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N,or 1O, thereof, wherein 1A is a methanol methyltransferase; 1B is amethylenetetrahydrofolate reductase; 1C is a methylenetetrahydrofolatedehydrogenase; 1D is a methenyltetrahydrofolate cyclohydrolase; 1E is aformyltetrahydrofolate deformylase; 1F is a formyltetrahydrofolatesynthetase; 1G is a formate hydrogen lyase; 1H is a hydrogenase, 1I is aformate dehydrogenase; 1J is a methanol dehydrogenase; 1K is spontaneousor formaldehyde activating enzyme; 1L is a formaldehyde dehydrogenase;1M is spontaneous or a S-(hydroxymethyl)glutathione synthase; 1N isglutathione-dependent formaldehyde dehydrogenase and 1O isS-formylglutathione hydrolase; and (2) the formaldehyde assimilationpathway comprises 3A, 3B or a combination thereof, wherein 3A is ahexylose-6-phosphate synthase, and 3B is a 6-phospho-3-hexyloisomerase.In some embodiments, 1K is spontaneous. In other embodiments, 1K is aformaldehyde activating enzyme. In some embodiments, 1M is spontaneous.In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. Insome embodiments, the intermediate is a hexylose-6-phosphate. In otherembodiments, the intermediate is a fructose-6-phosphate. In yet otherembodiments, the intermediate is a hexylose-6-phosphate and afructose-6-phosphate.

In one embodiment, the formaldehyde assimilation pathway comprises 3A.In another embodiment, the formaldehyde assimilation pathway comprises3B. In one embodiment, the formaldehyde assimilation pathway comprises3A and 3B.

In some embodiments, the methanol metabolic pathway is a methanolmetabolic pathway depicted in FIG. 1, and a formaldehyde assimilationpathway depicted in FIG. 3. An exemplary set of formaldehydeassimilation pathway enzymes to convert D-ribulose-5-phosphate andformaldehyde to fructose-6-phosphate (via hexylose-6-phosphate)according to FIG. 3 include 3A and 3B.

In a specific embodiment, (1) the methanol metabolic pathway comprises1J; and (2) the formaldehyde assimilation pathway comprises 3A and 3B.In other embodiments, (1) the methanol metabolic pathway comprises 1Jand 1K; and (2) the formaldehyde assimilation pathway comprises 3A and3B. In some embodiments, (1) the methanol metabolic pathway comprises1J, 1C, 1D and 1E; and (2) the formaldehyde assimilation pathwaycomprises 3A and 3B. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1C, 1D and 1F; and (2) the formaldehydeassimilation pathway comprises 3A and 3B. In another embodiment, (1) themethanol metabolic pathway comprises 1J and 1L; and (2) the formaldehydeassimilation pathway comprises 3A and 3B. In yet another embodiment, (1)the methanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2) theformaldehyde assimilation pathway comprises 3A and 3B. In certainembodiments, (1) the methanol metabolic pathway comprises 1J, 1N and 1O;and (2) the formaldehyde assimilation pathway comprises 3A and 3B. Insome embodiments, (1) the methanol metabolic pathway comprises 1J, 1K,1C, 1D and 1E; and (2) the formaldehyde assimilation pathway comprises3A and 3B. In one embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D and 1F; and (2) the formaldehyde assimilationpathway comprises 3A and 3B. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and (2) theformaldehyde assimilation pathway comprises 3A and 3B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1Fand 1I; and (2) the formaldehyde assimilation pathway comprises 3A and3B. In another embodiment, (1) the methanol metabolic pathway comprises1J, 1L and 1I; and (2) the formaldehyde assimilation pathway comprises3A and 3B. In yet another embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1M, 1N, 1O and 1I; and (2) the formaldehyde assimilationpathway comprises 3A and 3B. In certain embodiments, (1) the methanolmetabolic pathway comprises 1J, 1N, 1O and 1I; and (2) the formaldehydeassimilation pathway comprises 3A and 3B. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I; and (2)the formaldehyde assimilation pathway comprises 3A and 3B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F and 1I; and (2) the formaldehyde assimilation pathway comprises 3Aand 3B. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E and 1G; and (2) the formaldehyde assimilationpathway comprises 3A and 3B. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) theformaldehyde assimilation pathway comprises 3A and 3B. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G;and (2) the formaldehyde assimilation pathway comprises 3A and 3B. Inyet another embodiment, (1) the methanol metabolic pathway comprises 1J,1M, 1N, 1O and 1G; and (2) the formaldehyde assimilation pathwaycomprises 3A and 3B. In certain embodiments, (1) the methanol metabolicpathway comprises 1J, 1N, 1O and 1G; and (2) the formaldehydeassimilation pathway comprises 3A and 3B. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2)the formaldehyde assimilation pathway comprises 3A and 3B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F and 1G; and (2) the formaldehyde assimilation pathway comprises 3Aand 3B. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the formaldehydeassimilation pathway comprises 3A and 3B. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2)the formaldehyde assimilation pathway comprises 3A and 3B. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L, 1G and1H; and (2) the formaldehyde assimilation pathway comprises 3A and 3B.In yet another embodiment, (1) the methanol metabolic pathway comprises1J, 1M, 1N, 1O, 1G and 1H; and (2) the formaldehyde assimilation pathwaycomprises 3A and 3B. In certain embodiments, (1) the methanol metabolicpathway comprises 1J, 1N, 1O, 1G and 1H; and (2) the formaldehydeassimilation pathway comprises 3A and 3B. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and(2) the formaldehyde assimilation pathway comprises 3A and 3B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises3A and 3B. In some embodiments, 1K is spontaneous. In other embodiments,1K is a formaldehyde activating enzyme. In some embodiments, 1M isspontaneous. In some embodiments, the intermediate is ahexylose-6-phosphate. In other embodiments, the intermediate is afructose-6-phosphate. In yet other embodiments, the intermediate is ahexylose-6-phosphate and a fructose-6-phosphate.

In certain embodiments, (1) the methanol metabolic pathway comprises:1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or anycombination of 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N,or 1O, thereof, wherein 1A is a methanol methyltransferase; 1B is amethylenetetrahydrofolate reductase; 1C is a methylenetetrahydrofolatedehydrogenase; 1D is a methenyltetrahydrofolate cyclohydrolase; 1E is aformyltetrahydrofolate deformylase; 1F is a formyltetrahydrofolatesynthetase; 1G is a formate hydrogen lyase; 1H is a hydrogenase, 1I is aformate dehydrogenase; 1J is a methanol dehydrogenase; 1K is spontaneousor formaldehyde activating enzyme; 1L is a formaldehyde dehydrogenase;1M is spontaneous or a S-(hydroxymethyl)glutathione synthase; 1N isglutathione-dependent formaldehyde dehydrogenase and 1O isS-formylglutathione hydrolase; and (2) the formaldehyde assimilationpathway comprises 4A, 4B or a combination thereof, wherein 4A is adihydroxyacetone synthase and 4B is a dihydroxyacetone kinase. In someembodiments, 1K is spontaneous. In other embodiments, 1K is aformaldehyde activating enzyme. In some embodiments, 1M is spontaneous.In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. Insome embodiments, the intermediate is a DHA. In other embodiments, theintermediate is a dihydroxyacetone phosphate. In yet other embodiments,the intermediate is a DHA and a dihydroxyacetone phosphate.

In one embodiment, the formaldehyde assimilation pathway comprises 4A.In another embodiment, the formaldehyde assimilation pathway comprises4B. In one embodiment, the formaldehyde assimilation pathway comprises4A and 4B.

In some embodiments, the methanol metabolic pathway is a methanolmetabolic pathway depicted in FIG. 1, and a formaldehyde assimilationpathway depicted in FIG. 4. An exemplary set of formaldehydeassimilation pathway enzymes to convert xyulose-5-phosphate andformaldehyde to dihydroxyacetone-phosphate (via DHA) according to FIG. 4include 4A and 4B.

In a specific embodiment, (1) the methanol metabolic pathway comprises1J; and (2) the formaldehyde assimilation pathway comprises 4A and 4B.In other embodiments, (1) the methanol metabolic pathway comprises 1Jand 1K; and (2) the formaldehyde assimilation pathway comprises 4A and4B. In some embodiments, (1) the methanol metabolic pathway comprises1J, 1C, 1D and 1E; and (2) the formaldehyde assimilation pathwaycomprises 4A and 4B. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1C, 1D and 1F; and (2) the formaldehydeassimilation pathway comprises 4A and 4B. In another embodiment, (1) themethanol metabolic pathway comprises 1J and 1L; and (2) the formaldehydeassimilation pathway comprises 4A and 4B. In yet another embodiment, (1)the methanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2) theformaldehyde assimilation pathway comprises 4A and 4B. In certainembodiments, (1) the methanol metabolic pathway comprises 1J, 1N and 1O;and (2) the formaldehyde assimilation pathway comprises 4A and 4B. Insome embodiments, (1) the methanol metabolic pathway comprises 1J, 1K,1C, 1D and 1E; and (2) the formaldehyde assimilation pathway comprises4A and 4B. In one embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D and 1F; and (2) the formaldehyde assimilationpathway comprises 4A and 4B. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and (2) theformaldehyde assimilation pathway comprises 4A and 4B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1Fand 1I; and (2) the formaldehyde assimilation pathway comprises 4A and4B. In another embodiment, (1) the methanol metabolic pathway comprises1J, 1L and 1I; and (2) the formaldehyde assimilation pathway comprises4A and 4B. In yet another embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1M, 1N, 1O and 1I; and (2) the formaldehyde assimilationpathway comprises 4A and 4B. In certain embodiments, (1) the methanolmetabolic pathway comprises 1J, 1N, 1O and 1I; and (2) the formaldehydeassimilation pathway comprises 4A and 4B. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I; and (2)the formaldehyde assimilation pathway comprises 4A and 4B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F and 1I; and (2) the formaldehyde assimilation pathway comprises 4Aand 4B. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E and 1G; and (2) the formaldehyde assimilationpathway comprises 4A and 4B. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) theformaldehyde assimilation pathway comprises 4A and 4B. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G;and (2) the formaldehyde assimilation pathway comprises 4A and 4B. Inyet another embodiment, (1) the methanol metabolic pathway comprises 1J,1M, 1N, 1O and 1G; and (2) the formaldehyde assimilation pathwaycomprises 4A and 4B. In certain embodiments, (1) the methanol metabolicpathway comprises 1J, 1N, 1O and 1G; and (2) the formaldehydeassimilation pathway comprises 4A and 4B. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2)the formaldehyde assimilation pathway comprises 4A and 4B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F and 1G; and (2) the formaldehyde assimilation pathway comprises 4Aand 4B. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the formaldehydeassimilation pathway comprises 4A and 4B. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2)the formaldehyde assimilation pathway comprises 4A and 4B. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L, 1G and1H; and (2) the formaldehyde assimilation pathway comprises 4A and 4B.In yet another embodiment, (1) the methanol metabolic pathway comprises1J, 1M, 1N, 1O, 1G and 1H; and (2) the formaldehyde assimilation pathwaycomprises 4A and 4B. In certain embodiments, (1) the methanol metabolicpathway comprises 1J, 1N, 1O, 1G and 1H; and (2) the formaldehydeassimilation pathway comprises 4A and 4B. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and(2) the formaldehyde assimilation pathway comprises 4A and 4B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises4A and 4B. In some embodiments, 1K is spontaneous. In other embodiments,1K is a formaldehyde activating enzyme. In some embodiments, 1M isspontaneous. In some embodiments, the intermediate is a DHA. In otherembodiments, the intermediate is a dihydroxyacetone phosphate. In yetother embodiments, the intermediate is a DHA and a dihydroxyacetonephosphate.

Any methanol metabolic pathway provided herein can be combined with anyformaldehyde assimilation pathway provided herein. In addition, anymethanol metabolic pathway provided herein can be combined with anysuccinate pathway and any formaldehyde pathway provided herein.

Also provided herein are methods of producing formaldehyde comprisingculturing a non-naturally occurring microbial organism having a methanolmetabolic pathway provided herein. In some embodiments, the methanolmetabolic pathway comprises 1J. In certain embodiments, the organism iscultured in a substantially anaerobic culture medium. In specificembodiments, the formaldehyde is an intermediate that is consumed(assimilated) in the production of succinate and other productsdescribed herein.

Also provided herein are methods of producing an intermediate ofglycolysis and/or a metabolic pathway that can be used, for example, inthe formation of biomass, comprising culturing a non-naturally occurringmicrobial organism having a methanol metabolic pathway and aformaldehyde assimilation pathway, as provided herein, under conditionsand for a sufficient period of time to produce the intermediate. In someembodiments, the intermediate is a hexylose-6-phosphate. In otherembodiments, the intermediate is a fructose-6-phosphate. In yet otherembodiments, the intermediate is a hexylose-6-phosphate and afructose-6-phosphate. In some embodiments, the intermediate is a DHA. Inother embodiments, the intermediate is a dihydroxyacetone phosphate. Inyet other embodiments, the intermediate is a DHA and a dihydroxyacetonephosphate. In some embodiments, the methanol metabolic pathway comprises1J. In certain embodiments, the organism is cultured in a substantiallyanaerobic culture medium. Such biomass can also be used in methods ofproducing any of the products, such as the biobased products, providedelsewhere herein.

In certain embodiments, the organism comprises two, three, four or fivenucleic acids, each encoding a succinate pathway enzyme. In someembodiments, the organism comprises two nucleic acids, each encoding asuccinate pathway enzyme. In some embodiments, the organism comprisesthree nucleic acids, each encoding a succinate pathway enzyme. In someembodiments, the organism comprises four nucleic acids, each encoding asuccinate pathway enzyme. In other embodiments, the organism comprisesfive nucleic acids, each encoding a succinate pathway enzyme. In someembodiments, the nucleic acid encoding a succinate pathway enzyme is anexogenous nucleic acid. In other embodiments, the nucleic acid encodingan succinate pathway enzyme is an endogenous nucleic acid. In certainembodiments, the organism comprises two, three, four, five, six or sevennucleic acids, each encoding a succinate pathway enzyme; and theorganism further comprises two, three, four, five, six or sevenexogenous nucleic acids, each encoding a methanol metabolic pathwayenzyme. In certain embodiments, the organism further comprises twoexogenous nucleic acids, each encoding a methanol metabolic pathwayenzyme. In certain embodiments, the organism further comprises threeexogenous nucleic acids, each encoding a methanol metabolic pathwayenzyme. In certain embodiments, the organism comprises further fourexogenous nucleic acids, each encoding a methanol metabolic pathwayenzyme. In certain embodiments, the organism further comprises fiveexogenous nucleic acids, each encoding a methanol metabolic pathwayenzyme. In certain embodiments, the organism further comprises sixexogenous nucleic acids, each encoding a methanol metabolic pathwayenzyme. In certain embodiments, the organism further comprises sevenexogenous nucleic acids, each encoding a methanol metabolic pathwayenzyme.

In some embodiments, the organism comprises two or more exogenousnucleic acids, each encoding a formaldehyde assimilation pathway enzyme.In some embodiments, the organism comprises two exogenous nucleic acids,each encoding a formaldehyde assimilation pathway enzyme. In certainembodiments, the organism comprises two exogenous nucleic acids, eachencoding a formaldehyde assimilation pathway enzyme; and the organismfurther comprises two, three, four, five, six or seven exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism further comprises two exogenous nucleic acids,each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism further comprises three exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism comprises further four exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism further comprises five exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism further comprises six exogenous nucleic acids,each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism further comprises seven exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme.

In some embodiments, the at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme is a heterologous nucleic acid. Inother embodiments, the at least one exogenous nucleic acid encoding aformaldehyde assimilation pathway enzyme is a heterologous nucleic acid.In other embodiments, the at least one nucleic acid encoding a succinatepathway enzyme is a heterologous nucleic acid. In certain embodiments,the at least one exogenous nucleic acid encoding a methanol metabolicpathway enzyme is a heterologous nucleic acid, and the at least onenucleic acid encoding a succinate pathway enzyme is a heterologousnucleic acid. In other embodiments, the at least one exogenous nucleicacid encoding a methanol metabolic pathway enzyme is a heterologousnucleic acid, and the at least one exogenous nucleic acid encoding aformaldehyde assimilation pathway enzyme is a heterologous nucleic acid.

In certain embodiments, the organism is in a substantially anaerobicculture medium.

It is understood that any of the pathways disclosed herein, as describedin the Examples and exemplified in the figures, including the pathwaysof FIGS. 1, 2, 3 and 4, can be utilized to generate a non-naturallyoccurring microbial organism that produces any pathway intermediate orproduct, as desired. A non-limiting example of such an intermediate orproduct is succinate. As disclosed herein, such a microbial organismthat produces an intermediate can be used in combination with anothermicrobial organism expressing downstream pathway enzymes to produce adesired product. However, it is understood that a non-naturallyoccurring eukaryotic organism that produces a succinate pathwayintermediate can be utilized to produce the intermediate as a desiredproduct.

In certain embodiments, a non-naturally occurring microbial organismcomprising a methanol metabolic pathway and a succinate pathway providedherein, further comprises one or more gene disruptions. In certainembodiments, the one or more gene disruptions confer increasedproduction of succinate in the organism. In other embodiments, anon-naturally occurring microbial organism comprising a methanolmetabolic pathway and a formaldehyde assimilation pathway providedherein, further comprises one or more gene disruptions. In someembodiments, the gene disruption is in an endogenous gene encoding aprotein and/or enzyme involved in native production of ethanol,glycerol, acetate, lactate, formate, CO₂, amino acids, or anycombination thereof, by said microbial organism. In one embodiment, thegene disruption is in an endogenous gene encoding a protein and/orenzyme involved in native production of ethanol. In another embodiment,the gene disruption is in an endogenous gene encoding a protein and/orenzyme involved in native production of glycerol. In other embodiments,the gene disruption is in an endogenous gene encoding a protein and/orenzyme involved in native production of acetate. In another embodiment,the gene disruption is in an endogenous gene encoding a protein and/orenzyme involved in native production of lactate. In one embodiment, thegene disruption is in an endogenous gene encoding a protein and/orenzyme involved in native production of formate. In another embodiment,the gene disruption is in an endogenous gene encoding a protein and/orenzyme involved in native production of CO₂. In other embodiments, thegene disruption is in an endogenous gene encoding a protein and/orenzyme involved in native production of amino acids by said microbialorganism. The protein or enzyme is a pyruvate decarboxylase, an ethanoldehydrogenase, a glycerol dehydrogenase, a glycerol-3-phosphatase, aglycerol-3-phosphate dehydrogenase, a lactate dehydrogenase, an acetatekinase, a phosphotransacetylase, a pyruvate oxidase, a pyruvate:quinoneoxidoreductase, a pyruvate formate lyase, an alcohol dehydrogenase, alactate dehydrogenase, a pyruvate dehydrogenase, a pyruvateformate-lyase-2-ketobutyrate formate-lyase, a pyruvate transporter, amonocarboxylate transporter, a NADH dehydrogenase, a cytochrome oxidase,a pyruvate kinase, or any combination thereof. Non-limiting exemplarygenes encoding these proteins or enzymes are provided in Example IVbelow. In certain embodiments, the organism comprises from one totwenty-five gene disruptions. In other embodiments, the organismcomprises from one to twenty gene disruptions. In some embodiments, theorganism comprises from one to fifteen gene disrutions. In otherembodiments, the organism comprises from one to ten gene disruptions. Insome embodiments, the organism comprises from one to five genedisruptions. In certain embodiments, the organism comprises 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24 or 25 gene disruptions or more.

In other embodiments, a non-naturally occurring microbial organismcomprising a methanol metabolic pathway and a succinate pathway providedherein, further comprises one or more endogenous proteins or enzymesinvolved in native production of ethanol, glycerol, acetate, lactate,formate, CO₂ and/or amino acids by said microbial organism, wherein saidone or more endogenous proteins or enzymes has attenuated protein orenzyme activity and/or expression levels. In some embodiments, anon-naturally occurring microbial organism comprising a methanolmetabolic pathway and a formaldehyde assimilation pathway providedherein, further comprises one or more endogenous proteins or enzymesinvolved in native production of ethanol, glycerol, acetate, lactate,formate, CO2 and/or amino acids by said microbial organism, wherein saidone or more endogenous proteins or enzymes has attenuated protein orenzyme activity and/or expression levels. In one embodiment theendogenous protein or enzyme is a pyruvate decarboxylase, an ethanoldehydrogenase, a glycerol dehydrogenase, a glycerol-3-phosphatase, aglycerol-3-phosphate dehydrogenase, a lactate dehydrogenase, an acetatekinase, a phosphotransacetylase, a pyruvate oxidase, a pyruvate:quinoneoxidoreductase, a pyruvate formate lyase, an alcohol dehydrogenase, alactate dehydrogenase, a pyruvate dehydrogenase, a pyruvateformate-lyase-2-ketobutyrate formate-lyase, a pyruvate transporter, amonocarboxylate transporter, a NADH dehydrogenase, a cytochrome oxidase,a pyruvate kinase, or any combination thereof. Non-limiting exemplarygenes encoding these proteins or enzymes are provided in Example IVbelow.

Each of the non-naturally occurring alterations provided herein (see,e.g., Example IV) result in increased production and an enhanced levelof succinate, for example, during the exponential growth phase of themicrobial organism, compared to a strain that does not contain suchmetabolic alterations, under appropriate culture conditions. Appropriateconditions include, for example, those disclosed herein, includingconditions such as particular carbon sources or reactant availabilitiesand/or adaptive evolution.

Given the teachings and guidance provided herein (see, e.g., ExampleIV), those skilled in the art will understand that to introduce ametabolic alteration, such as attenuation of an enzyme, it can benecessary to disrupt the catalytic activity of the one or more enzymesinvolved in the reaction. Alternatively, a metabolic alteration caninclude disrupting expression of a regulatory protein or cofactornecessary for enzyme activity or maximal activity. Furthermore, geneticloss of a cofactor necessary for an enzymatic reaction can also have thesame effect as a disruption of the gene encoding the enzyme. Disruptioncan occur by a variety of methods including, for example, deletion of anencoding gene or incorporation of a genetic alteration in one or more ofthe encoding gene sequences. The encoding genes targeted for disruptioncan be one, some, or all of the genes encoding enzymes involved in thecatalytic activity. For example, where a single enzyme is involved in atargeted catalytic activity, disruption can occur by a geneticalteration that reduces or eliminates the catalytic activity of theencoded gene product. Similarly, where the single enzyme is multimeric,including heteromeric, disruption can occur by a genetic alteration thatreduces or destroys the function of one or all subunits of the encodedgene products. Destruction of activity can be accomplished by loss ofthe binding activity of one or more subunits required to form an activecomplex, by destruction of the catalytic subunit of the multimericcomplex or by both. Other functions of multimeric protein associationand activity also can be targeted in order to disrupt a metabolicreaction of the invention. Such other functions are well known to thoseskilled in the art. Similarly, a target enzyme activity can be reducedor eliminated by disrupting expression of a protein or enzyme thatmodifies and/or activates the target enzyme, for example, a moleculerequired to convert an apoenzyme to a holoenzyme. Further, some or allof the functions of a single polypeptide or multimeric complex can bedisrupted according to the invention in order to reduce or abolish thecatalytic activity of one or more enzymes involved in a reaction ormetabolic modification of the invention. Similarly, some or all ofenzymes involved in a reaction or metabolic modification of theinvention can be disrupted so long as the targeted reaction is reducedor eliminated.

Given the teachings and guidance provided herein (see, e.g., ExampleIV), those skilled in the art also will understand that an enzymaticreaction can be disrupted by reducing or eliminating reactions encodedby a common gene and/or by one or more orthologs of that gene exhibitingsimilar or substantially the same activity. Reduction of both the commongene and all orthologs can lead to complete abolishment of any catalyticactivity of a targeted reaction. However, disruption of either thecommon gene or one or more orthologs can lead to a reduction in thecatalytic activity of the targeted reaction sufficient to promotecoupling of growth to product biosynthesis. Exemplified herein are boththe common genes encoding catalytic activities for a variety ofmetabolic modifications as well as their orthologs. Those skilled in theart will understand that disruption of some or all of the genes encodinga enzyme of a targeted metabolic reaction can be practiced in themethods of the invention and incorporated into the non-naturallyoccurring microbial organisms of the invention in order to achieve theincreased production of fatty alcohol, fatty aldehyde or fatty acid orgrowth-coupled product production.

Given the teachings and guidance provided herein (see, e.g., ExampleIV), those skilled in the art also will understand that enzymaticactivity or expression can be attenuated using well known methods.Reduction of the activity or amount of an enzyme can mimic completedisruption if the reduction causes activity of the enzyme to fall belowa critical level that is normally required for the pathway to function.Reduction of enzymatic activity by various techniques rather thandisruption can be important for an organism's viability. Methods ofreducing enzymatic activity that result in similar or identical effectsof a gene disruption include, but are not limited to: reducing genetranscription or translation; destabilizing mRNA, protein or catalyticRNA; and mutating a gene that affects enzyme kinetics. Natural orimposed regulatory controls can also accomplish enzyme attenuationincluding: promoter replacement; loss or alteration of transcriptionfactors; introduction of inhibitory RNAs or peptides such as siRNA,antisense RNA, RNA or peptide/small-molecule binding aptamers,ribozymes, aptazymes and riboswitches; and addition of drugs and otherchemicals that reduce or disrupt enzymatic activity such as genesplicing.

One of ordinary skill in the art will also recognize that attenuation ofan enzyme (e.g., as provided in Example IV) can be done at variouslevels. For example, at the gene level, mutations causing a partial orcomplete null phenotype or epistatic genetic effects that mask theactivity of a gene product can be used to attenuate an enzyme. At thegene expression level, methods for attenuation include: couplingtranscription to an endogenous or exogenous inducer such as IPTG, thenadding low or 0 levels of inducer during the production phase;introducing or modifying positive or negative regulators; modify histoneacetylation/deacetylation in region where gene is integrated;introducing a transposition to disrupt a promoter or a regulatory gene;flipping of a transposable element or promoter region; deleting oneallele resulting in loss of heterozygosity in a diploid organism;introducing nucleic acids that increase RNA degradation; or in bacteria,for example, introduction of a tmRNA tag, which can lead to RNAdegradation and ribosomal stalling. At the translational level,attenuation can include: introducing rare codons to limit translation;introducing RNA interference molecules that block translation; modifyingregions outside the coding sequence, such as introducing secondarystructure into UTR regions to block translation or reduce efficiency oftranslation; adding RNAase sites for rapid transcript degradation;introducing antisense RNA oligomers or antisense transcripts;introducing RNA or peptide aptamers, ribozymes, aptazymes, riboswitches;or introducing translational regulatory elements involving RNA structurethat can prevent or reduce translation that can be controlled by thepresence or absence of small molecules. At the level of enzymelocalization and/or longevity, enzyme attenuation can include: adding adegradation tag for faster protein turnover; or adding a localizationtag that results in the enzyme being localized to a compartment where itwould not be able to react normally. At the level of post-translationalregulation, enzyme attenuation can include: increasing intracellularconcentration of known inhibitors; or modifying post-translationalmodified sites. At the level of enzyme activity, enzyme attenuation caninclude: adding endogenous or exogenous inhibitor, such as atarget-specific drug, to reduce enzyme activity; limiting availabilityof essential cofactors, such as B12, for an enzyme that require it;chelating a metal ion that is required for activity; or introducing adominant negative mutation.

In some embodiments, microaerobic designs can be used based on thegrowth-coupled formation of the desired product. To examine this,production cones can be constructed for each strategy by firstmaximizing and, subsequently minimizing the product yields at differentrates of biomass formation feasible in the network. If the rightmostboundary of all possible phenotypes of the mutant network is a singlepoint, it implies that there is a unique optimum yield of the product atthe maximum biomass formation rate possible in the network. In othercases, the rightmost boundary of the feasible phenotypes is a verticalline, indicating that at the point of maximum biomass the network canmake any amount of the product in the calculated range, including thelowest amount at the bottommost point of the vertical line. Such designsare given a low priority.

The succinate-production strategies identified in the various tablesdisclosed herein (e.g., Example IV) can be disrupted to increaseproduction of succinate. Accordingly, also provided herein is anon-naturally occurring microbial organism having metabolicmodifications coupling succinate production to growth of the organism,where the metabolic modifications includes disruption of one or moregenes selected from the genes encoding proteins and/or enzymes shown inthe various tables disclosed herein.

Each of the strains can be supplemented with additional deletions if itis determined that the strain designs do not sufficiently increase theproduction of succinate and/or couple the formation of the product withbiomass formation. Alternatively, some other enzymes not known topossess significant activity under the growth conditions can becomeactive due to adaptive evolution or random mutagenesis. Such activitiescan also be knocked out. However, the list of gene deletion disclosedherein allows the construction of strains exhibiting high-yieldproduction of succinate, including growth-coupled production ofsuccinate.

In another aspect, provided herein is a method for producing succinate,comprising culturing any one of the non-naturally occurring microbialorganisms comprising a methanol metabolic pathway and a succinatepathway provided herein under conditions and for a sufficient period oftime to produce succinate. In certain embodiments, the organism iscultured in a substantially anaerobic culture medium.

In one embodiment, provided herein are methods for producing succinate,comprising culturing an organism provided herein (e.g., a non-naturallyoccurring microbial organisms comprising a methanol metabolic pathwayand a succinate pathway) under conditions and for a sufficient period oftime to produce succinate. In some embodiments, the method comprisesculturing, for a sufficient period of time to produce succinate, anon-naturally occurring microbial organism, comprising (1) a methanolmetabolic pathway, wherein said organism comprises at least oneexogenous nucleic acid encoding a methanol metabolic pathway enzyme in asufficient amount to enhance the availability of reducing equivalents inthe presence of methanol; and (2) a succinate pathway.

In certain embodiments of the methods provided herein, the organismfurther comprises at least one nucleic acid encoding a succinate pathwayenzyme expressed in a sufficient amount to produce succinate. In someembodiments, the nucleic acid encoding a succinate pathway enzyme is anexogenous nucleic acid. In other embodiments, the nucleic acid encodingan succinate pathway enzyme is an endogenous nucleic acid. In someembodiments, the organism further comprises one or more gene disruptionsprovided herein that confer increased production of succinate in theorganism. In certain embodiments, the one or more gene disruptionsoccurs in an endogenous gene encoding a protein or enzyme involved innative production of ethanol, glycerol, acetate, lactate, formate, CO₂and/or amino acids by said microbial organism. In other embodiments, theorganism further comprises one or more endogenous proteins or enzymesinvolved in native production of ethanol, glycerol, acetate, lactate,formate, CO₂ and/or amino acids by said microbial organism, wherein saidone or more endogenous proteins or enzymes has attenuated protein orenzyme activity and/or expression levels. In certain embodiments, theorganism is a Crabtree positive, eukaryotic organism, and the organismis cultured in a culture medium comprising glucose. In certainembodiments, the organism comprises from one to twenty-five genedisruptions. In other embodiments, the organism comprises from one totwenty gene disruptions. In some embodiments, the organism comprisesfrom one to fifteen gene disrutions. In other embodiments, the organismcomprises from one to ten gene disruptions. In some embodiments, theorganism comprises from one to five gene disruptions. In certainembodiments, the organism comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 genedisruptions or more.

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a succinate pathway, formaldehydeassimilation pathway and/or methanol metabolic pathway, wherein thenon-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding an enzyme or protein that converts asubstrate to a product. By way of example, in FIG. 1, the substrate of1J is methanol, and the product is formaldehyde; the substrate of 1L isformaldehyde, and the product is formate; and so forth. One skilled inthe art will understand that these are merely exemplary and that any ofthe substrate-product pairs disclosed herein suitable to produce adesired product and for which an appropriate activity is available forthe conversion of the substrate to the product can be readily determinedby one skilled in the art based on the teachings herein. Thus, providedherein are non-naturally occurring microbial organisms containing atleast one exogenous nucleic acid encoding an enzyme or protein, wherethe enzyme or protein converts the substrates and products of a methanolmetabolic pathway, such as that shown in FIG. 1; a succinate pathway,such as that shown in FIG. 2; and/or a formaldehyde assimilationpathway, such as that shown in FIG. 3 or 4.

While generally described herein as a microbial organism that contains asuccinate pathway, formaldehyde assimilation pathway, and/or a methanolmetabolic pathway, it is understood that provided herein are alsonon-naturally occurring microbial organism comprising at least onenucleic acid encoding a succinate pathway, formaldehyde assimilationpathway, and/or a methanol metabolic pathway enzyme expressed in asufficient amount to produce an intermediate of a succinate pathway,formaldehyde assimilation pathway, and/or a methanol metabolic pathwayintermediate. For example, as disclosed herein, a succinate pathway isexemplified in FIG. 2. Therefore, in addition to a microbial organismcontaining a succinate pathway that produces succinate, also providedherein is a non-naturally occurring microbial organism comprising atleast one nucleic acid encoding a succinate pathway enzyme, where themicrobial organism produces a succinate pathway intermediate. In someembodiments, the nucleic acid encoding a succinate pathway enzyme is anexogenous nucleic acid. In other embodiments, the nucleic acid encodingan succinate pathway enzyme is an endogenous nucleic acid.

In some embodiments, the carbon feedstock and other cellular uptakesources such as phosphate, ammonia, sulfate, chloride and other halogenscan be chosen to alter the isotopic distribution of the atoms present insuccinate or any succinate pathway intermediate. The various carbonfeedstock and other uptake sources enumerated above will be referred toherein, collectively, as “uptake sources.” Uptake sources can provideisotopic enrichment for any atom present in the product succinate,and/or succinate pathway intermediate, or for side products generated inreactions diverging away from a succinate pathway. Isotopic enrichmentcan be achieved for any target atom including, for example, carbon,hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or otherhalogens.

In some embodiments, the uptake sources can be selected to alter thecarbon-12, carbon-13, and carbon-14 ratios. In some embodiments, theuptake sources can be selected to alter the oxygen-16, oxygen-17, andoxygen-18 ratios. In some embodiments, the uptake sources can beselected to alter the hydrogen, deuterium, and tritium ratios. In someembodiments, the uptake sources can selected to alter the nitrogen-14and nitrogen-15 ratios. In some embodiments, the uptake sources can beselected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35ratios. In some embodiments, the uptake sources can be selected to alterthe phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In someembodiments, the uptake sources can be selected to alter thechlorine-35, chlorine-36, and chlorine-37 ratios.

In some embodiments, the isotopic ratio of a target atom can be variedto a desired ratio by selecting one or more uptake sources. An uptakesource can be derived from a natural source, as found in nature, or froma man-made source, and one skilled in the art can select a naturalsource, a man-made source, or a combination thereof, to achieve adesired isotopic ratio of a target atom. An example of a man-made uptakesource includes, for example, an uptake source that is at leastpartially derived from a chemical synthetic reaction. Such isotopicallyenriched uptake sources can be purchased commercially or prepared in thelaboratory and/or optionally mixed with a natural source of the uptakesource to achieve a desired isotopic ratio. In some embodiments, atarget isotopic ratio of an uptake source can be obtained by selecting adesired origin of the uptake source as found in nature For example, asdiscussed herein, a natural source can be a biobased derived from orsynthesized by a biological organism or a source such as petroleum-basedproducts or the atmosphere. In some such embodiments, a source ofcarbon, for example, can be selected from a fossil fuel-derived carbonsource, which can be relatively depleted of carbon-14, or anenvironmental or atmospheric carbon source, such as CO₂, which canpossess a larger amount of carbon-14 than its petroleum-derivedcounterpart.

Isotopic enrichment is readily assessed by mass spectrometry usingtechniques known in the art such as Stable Isotope Ratio MassSpectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation byNuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques canbe integrated with separation techniques such as liquid chromatography(LC) and/or high performance liquid chromatography (HPLC).

The unstable carbon isotope carbon-14 or radiocarbon makes up forroughly 1 in 10¹² carbon atoms in the earth's atmosphere and has ahalf-life of about 5700 years. The stock of carbon is replenished in theupper atmosphere by a nuclear reaction involving cosmic rays andordinary nitrogen (¹⁴N). Fossil fuels contain no carbon-14, as itdecayed long ago. Burning of fossil fuels lowers the atmosphericcarbon-14 fraction, the so-called “Suess effect”.

Methods of determining the isotopic ratios of atoms in a compound arewell known to those skilled in the art. Isotopic enrichment is readilyassessed by mass spectrometry using techniques known in the art such asaccelerated mass spectrometry (AMS), Stable Isotope Ratio MassSpectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation byNuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques canbe integrated with separation techniques such as liquid chromatography(LC), high performance liquid chromatography (HPLC) and/or gaschromatography, and the like.

In the case of carbon, ASTM D6866 was developed in the United States asa standardized analytical method for determining the biobased content ofsolid, liquid, and gaseous samples using radiocarbon dating by theAmerican Society for Testing and Materials (ASTM) International. Thestandard is based on the use of radiocarbon dating for the determinationof a product's biobased content. ASTM D6866 was first published in 2004,and the current active version of the standard is ASTM D6866-11(effective Apr. 1, 2011). Radiocarbon dating techniques are well knownto those skilled in the art, including those described herein.

The biobased content of a compound is estimated by the ratio ofcarbon-14 (¹⁴C) to carbon-12 (¹²C). Specifically, the Fraction Modern(Fm) is computed from the expression: Fm=(S−B)/(M−B), where B, S and Mrepresent the ¹⁴C/¹²C ratios of the blank, the sample and the modernreference, respectively. Fraction Modern is a measurement of thedeviation of the ¹⁴C/¹²C ratio of a sample from “Modern.” Modern isdefined as 95% of the radiocarbon concentration (in AD 1950) of NationalBureau of Standards (NBS) Oxalic Acid I (i.e., standard referencematerials (SRM) 4990b) normalized to δ¹³C_(VPDB)=−19 per mil (Olsson,The use of Oxalic acid as a Standard. in, Radiocarbon Variations andAbsolute Chronology, Nobel Symposium, 12th Proc., John Wiley & Sons, NewYork (1970)). Mass spectrometry results, for example, measured by ASM,are calculated using the internationally agreed upon definition of 0.95times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalizedto δ¹³C_(VPDB)=−19 per mil. This is equivalent to an absolute (AD 1950)¹⁴C/¹²C ratio of 1.176±0.010×10⁻¹² (Karlen et al., Arkiv Geofysik,4:465-471 (1968)). The standard calculations take into account thedifferential uptake of one isotope with respect to another, for example,the preferential uptake in biological systems of C¹² over C¹³ over C¹⁴,and these corrections are reflected as a Fm corrected for δ¹³.

An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of1955 sugar beet. Although there were 1000 lbs made, this oxalic acidstandard is no longer commercially available. The Oxalic Acid IIstandard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of1977 French beet molasses. In the early 1980's, a group of 12laboratories measured the ratios of the two standards. The ratio of theactivity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). Theisotopic ratio of HOx II is −17.8 per mille. ASTM D6866-11 suggests useof the available Oxalic Acid II standard SRM 4990 C (Hox2) for themodern standard (see discussion of original vs. currently availableoxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)). AFm=0% represents the entire lack of carbon-14 atoms in a material, thusindicating a fossil (for example, petroleum based) carbon source. AFm=100%, after correction for the post-1950 injection of carbon-14 intothe atmosphere from nuclear bomb testing, indicates an entirely moderncarbon source. As described herein, such a “modern” source includesbiobased sources.

As described in ASTM D6866, the percent modern carbon (pMC) can begreater than 100% because of the continuing but diminishing effects ofthe 1950s nuclear testing programs, which resulted in a considerableenrichment of carbon-14 in the atmosphere as described in ASTM D6866-11.Because all sample carbon-14 activities are referenced to a “pre-bomb”standard, and because nearly all new biobased products are produced in apost-bomb environment, all pMC values (after correction for isotopicfraction) must be multiplied by 0.95 (as of 2010) to better reflect thetrue biobased content of the sample. A biobased content that is greaterthan 103% suggests that either an analytical error has occurred, or thatthe source of biobased carbon is more than several years old.

ASTM D6866 quantifies the biobased content relative to the material'stotal organic content and does not consider the inorganic carbon andother non-carbon containing substances present. For example, a productthat is 50% starch-based material and 50% water would be considered tohave a Biobased Content=100% (50% organic content that is 100% biobased)based on ASTM D6866. In another example, a product that is 50%starch-based material, 25% petroleum-based, and 25% water would have aBiobased Content=66.7% (75% organic content but only 50% of the productis biobased). In another example, a product that is 50% organic carbonand is a petroleum-based product would be considered to have a BiobasedContent=0% (50% organic carbon but from fossil sources). Thus, based onthe well known methods and known standards for determining the biobasedcontent of a compound or material, one skilled in the art can readilydetermine the biobased content and/or prepared downstream products thatutilize of the invention having a desired biobased content.

Applications of carbon-14 dating techniques to quantify bio-basedcontent of materials are known in the art (Currie et al., NuclearInstruments and Methods in Physics Research B, 172:281-287 (2000)). Forexample, carbon-14 dating has been used to quantify bio-based content interephthalate-containing materials (Colonna et al., Green Chemistry,13:2543-2548 (2011)). Notably, polypropylene terephthalate (PPT)polymers derived from renewable 1,3-propanediol and petroleum-derivedterephthalic acid resulted in Fm values near 30% (i.e., since 3/11 ofthe polymeric carbon derives from renewable 1,3-propanediol and 8/11from the fossil end member terephthalic acid) (Currie et al., supra,2000). In contrast, polybutylene terephthalate polymer derived from bothrenewable BDO and renewable terephthalic acid resulted in bio-basedcontent exceeding 90% (Colonna et al., supra, 2011).

Accordingly, in some embodiments, the present invention providessuccinate, or a succinate pathway intermediate thereof, that has acarbon-12, carbon-13, and carbon-14 ratio that reflects an atmosphericcarbon, also referred to as environmental carbon, uptake source. Forexample, in some aspects, the succinate, or a succinate intermediatethereof can have an Fm value of at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98% or as much as 100%. In some such embodiments, theuptake source is CO₂. In some embodiments, the present inventionprovides succinate, or a succinate intermediate thereof, that has acarbon-12, carbon-13, and carbon-14 ratio that reflects petroleum-basedcarbon uptake source. In this aspect, the a succinate, or a succinateintermediate can have an Fm value of less than 95%, less than 90%, lessthan 85%, less than 80%, less than 75%, less than 70%, less than 65%,less than 60%, less than 55%, less than 50%, less than 45%, less than40%, less than 35%, less than 30%, less than 25%, less than 20%, lessthan 15%, less than 10%, less than 5%, less than 2% or less than 1%. Insome embodiments, the present invention provides a succinate, or asuccinate intermediate thereof, that has a carbon-12, carbon-13, andcarbon-14 ratio that is obtained by a combination of an atmosphericcarbon uptake source with a petroleum-based uptake source. Using such acombination of uptake sources is one way by which the carbon-12,carbon-13, and carbon-14 ratio can be varied, and the respective ratioswould reflect the proportions of the uptake sources.

Further, the present invention relates to biologically producedsuccinate, or a succinate intermediate thereof, as disclosed herein, andto the products derived therefrom, wherein the a succinate, or anintermediate thereof, has a carbon-12, carbon-13, and carbon-14 isotoperatio of about the same value as the CO₂ that occurs in the environment.For example, in some aspects the invention provides bioderivedsuccinate, or an intermediate thereof, having a carbon-12 versuscarbon-13 versus carbon-14 isotope ratio of about the same value as theCO₂ that occurs in the environment, or any of the other ratios disclosedherein. It is understood, as disclosed herein, that a product can have acarbon-12 versus carbon-13 versus carbon-14 isotope ratio of about thesame value as the CO₂ that occurs in the environment, or any of theratios disclosed herein, wherein the product is generated frombioderived succinate, or an intermediate thereof, as disclosed herein,wherein the bioderived product is chemically modified to generate afinal product. Methods of chemically modifying a bioderived product ofsuccinate, or an intermediate thereof, to generate a desired product arewell known to those skilled in the art, as described herein. Theinvention further provides products made or derived from succinate,including but not limited to butanediol, tetrahydrofuran, pyrrolidone,solvents, paints, deicers, plastics, fuel additives, fabrics, carpets,pigments, detergents, metal plating; polymers such as polybutylenesuccinate polymers, which can be used as a biodegradable plastic toreplace conventional plastics in applications such as flexiblepackaging, agricultural films and compostable bags; a combination ofpolybutylene succinate with polymers such as polypropylene (PP),polystyrene (PS) and polycarbonate (PC), and with plastics such aspolylactic acid, polyhydroxyalkanoate, and poly-3-hydroxybutyrateco-valerate; and a combination of polybutylene succinate withfibers or fillers for applications such as automotive interiors,nonwovens, construction materials and consumer goods, and the like,having a carbon-12 versus carbon-13 versus carbon-14 isotope ratio ofabout the same value as the CO₂ that occurs in the environment, whereinthe invention further provides products made or derived from succinate,including but not limited to butanediol, tetrahydrofuran, pyrrolidone,solvents, paints, deicers, plastics, fuel additives, fabrics, carpets,pigments, detergents, metal plating; polymers such as polybutylenesuccinate polymers, which can be used as a biodegradable plastic toreplace conventional plastics in applications such as flexiblepackaging, agricultural films and compostable bags; a combination ofpolybutylene succinate with polymers such as PP, PS and PC, and withplastics such as polylactic acid, polyhydroxyalkanoate, andpoly-3-hydroxy butyrateco-valerate; and a combination of polybutylenesuccinate with fibers or fillers for applications such as automotiveinteriors, nonwovens, construction materials and consumer goods, and thelike, are generated directly from or in combination with bioderivedsuccinate or a bioderived intermediate thereof, as disclosed herein.

Succinate, as well as intermediates thereof, are chemicals used incommercial and industrial applications. Non-limiting examples of suchapplications include production of butanediol, tetrahydrofuran,pyrrolidone, solvents, paints, deicers, plastics, fuel additives,fabrics, carpets, pigments, detergents, metal plating; polymers such aspolybutylene succinate polymers, which can be used as a biodegradableplastic to replace conventional plastics in applications such asflexible packaging, agricultural films and compostable bags; acombination of polybutylene succinate with polymers such as PP, PS andPC, and with plastics such as polylactic acid, polyhydroxyalkanoate, andpoly-3-hydroxy butyrateco-valerate; and a combination of polybutylenesuccinate with fibers or fillers for applications such as automotiveinteriors, nonwovens, construction materials and consumer goods, and thelike. Moreover, succinate are also used as a raw material in theproduction of a wide range of products including butanediol,tetrahydrofuran, pyrrolidone, solvents, paints, deicers, plastics, fueladditives, fabrics, carpets, pigments, detergents, metal plating;polymers such as polybutylene succinate polymers, which can be used as abiodegradable plastic to replace conventional plastics in applicationssuch as flexible packaging, agricultural films and compostable bags; acombination of polybutylene succinate with polymers such as PP, PS andPC, and with plastics such as polylactic acid, polyhydroxyalkanoate, andpoly-3-hydroxy butyrateco-valerate; and a combination of polybutylenesuccinate with fibers or fillers for applications such as automotiveinteriors, nonwovens, construction materials and consumer goods, and thelike. Accordingly, in some embodiments, the invention provides biobasedbutanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers,plastics, fuel additives, fabrics, carpets, pigments, detergents, metalplating; polymers such as polybutylene succinate polymers, which can beused as a biodegradable plastic to replace conventional plastics inapplications such as flexible packaging, agricultural films andcompostable bags; a combination of polybutylene succinate with polymerssuch as PP, PS and PC, and with plastics such as polylactic acid,polyhydroxyalkanoate, and poly-3-hydroxy butyrateco-valerate; and acombination of polybutylene succinate with fibers or fillers forapplications such as automotive interiors, nonwovens, constructionmaterials and consumer goods, and the like, comprising one or more ofbioderived succinate, or a bioderived intermediate thereof, produced bya non-naturally occurring microorganism of the invention or producedusing a method disclosed herein.

As used herein, the term “bioderived” means derived from or synthesizedby a biological organism and can be considered a renewable resourcesince it can be generated by a biological organism. Such a biologicalorganism, in particular the microbial organisms of the inventiondisclosed herein, can utilize feedstock or biomass, such as, sugars orcarbohydrates obtained from an agricultural, plant, bacterial, or animalsource. Alternatively, the biological organism can utilize atmosphericcarbon. As used herein, the term “biobased” means a product as describedabove that is composed, in whole or in part, of a bioderived compound ofthe invention. A biobased or bioderived product is in contrast to apetroleum derived product, wherein such a product is derived from orsynthesized from petroleum or a petrochemical feedstock.

In some embodiments, the invention provides butanediol, tetrahydrofuran,pyrrolidone, solvents, paints, deicers, plastics, fuel additives,fabrics, carpets, pigments, detergents, metal plating; polymers such aspolybutylene succinate polymers, which can be used as a biodegradableplastic to replace conventional plastics in applications such asflexible packaging, agricultural films and compostable bags; acombination of polybutylene succinate with polymers such as PP, PS andPC, and with plastics such as polylactic acid, polyhydroxyalkanoate, andpoly-3-hydroxy butyrateco-valerate; and a combination of polybutylenesuccinate with fibers or fillers for applications such as automotiveinteriors, nonwovens, construction materials and consumer goods, and thelike, comprising bioderived succinate, or a bioderived intermediatethereof, wherein the bioderived succinate, or bioderived intermediatethereof, includes all or part of the a succinate, or an intermediatethereof, used in the production of butanediol, tetrahydrofuran,pyrrolidone, solvents, paints, deicers, plastics, fuel additives,fabrics, carpets, pigments, detergents, metal plating; polymers such aspolybutylene succinate polymers, which can be used as a biodegradableplastic to replace conventional plastics in applications such asflexible packaging, agricultural films and compostable bags; acombination of polybutylene succinate with polymers such as PP, PS andPC, and with plastics such as polylactic acid, polyhydroxyalkanoate, andpoly-3-hydroxy butyrateco-valerate; and a combination of polybutylenesuccinate with fibers or fillers for applications such as automotiveinteriors, nonwovens, construction materials and consumer goods, and thelike. Thus, in some aspects, the invention provides a biobasedbutanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers,plastics, fuel additives, fabrics, carpets, pigments, detergents, metalplating; polymers such as polybutylene succinate polymers, which can beused as a biodegradable plastic to replace conventional plastics inapplications such as flexible packaging, agricultural films andcompostable bags; a combination of polybutylene succinate with polymerssuch as PP, PS and PC, and with plastics such as polylactic acid,polyhydroxyalkanoate, and poly-3-hydroxy butyrateco-valerate; and acombination of polybutylene succinate with fibers or fillers forapplications such as automotive interiors, nonwovens, constructionmaterials and consumer goods, and the like, comprising at least 2%, atleast 3%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98% or 100% bioderived succinate, or a bioderived succinateintermediate, as disclosed herein. Additionally, in some aspects, theinvention provides biobased butanediol, tetrahydrofuran, pyrrolidone,solvents, paints, deicers, plastics, fuel additives, fabrics, carpets,pigments, detergents, metal plating; polymers such as polybutylenesuccinate polymers, which can be used as a biodegradable plastic toreplace conventional plastics in applications such as flexiblepackaging, agricultural films and compostable bags; a combination ofpolybutylene succinate with polymers such as PP, PS and PC, and withplastics such as polylactic acid, polyhydroxyalkanoate, andpoly-3-hydroxy butyrateco-valerate; and a combination of polybutylenesuccinate with fibers or fillers for applications such as automotiveinteriors, nonwovens, construction materials and consumer goods, and thelike, wherein the a succinate, or a succinate intermediate, used in itsproduction is a combination of bioderived and petroleum derivedsuccinate, or a succinate intermediate thereof. For example, biobasedbutanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers,plastics, fuel additives, fabrics, carpets, pigments, detergents, metalplating; polymers such as polybutylene succinate polymers, which can beused as a biodegradable plastic to replace conventional plastics inapplications such as flexible packaging, agricultural films andcompostable bags; a combination of polybutylene succinate with polymerssuch as PP, PS and PC, and with plastics such as polylactic acid,polyhydroxyalkanoate, and poly-3-hydroxy butyrateco-valerate; and acombination of polybutylene succinate with fibers or fillers forapplications such as automotive interiors, nonwovens, constructionmaterials and consumer goods, and the like, can be produced using 50%bioderived succinate and 50% petroleum derived succinate or otherdesired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%,100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleumderived precursors, so long as at least a portion of the productcomprises a bioderived product produced by the microbial organismsdisclosed herein. It is understood that methods for producingbutanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers,plastics, fuel additives, fabrics, carpets, pigments, detergents, metalplating; polymers such as polybutylene succinate polymers, which can beused as a biodegradable plastic to replace conventional plastics inapplications such as flexible packaging, agricultural films andcompostable bags; a combination of polybutylene succinate with polymerssuch as PP, PS and PC, and with plastics such as polylactic acid,polyhydroxyalkanoate, and poly-3-hydroxy butyrateco-valerate; and acombination of polybutylene succinate with fibers or fillers forapplications such as automotive interiors, nonwovens, constructionmaterials and consumer goods, and the like, using the bioderivedsuccinate, or a bioderived succinate intermediate thereof, of theinvention are well known in the art.

In one embodiment, the product is a butanediol. In one embodiment, theproduct is a tetrahydrofuran. In one embodiment, the product is apyrrolidone. In one embodiment, the product is a solvent. In oneembodiment, the product is a paint. In one embodiment, the product is adeicer. In one embodiment, the product is a plastic. In one embodiment,the product is a fuel additive. In one embodiment, the product is afabric. In one embodiment, the product is a carpet. In one embodiment,the product is a pigment. In one embodiment, the product is a detergent.In one embodiment, the product is a metal plating. In one embodiment,the product is a polymer. In one embodiment, the product is apolybutylene succinate polymer. In one embodiment, the product is abiodegradable plastic. In one embodiment, the product is a flexiblepackaging. In one embodiment, the product is an agricultural film. Inone embodiment, the product is a compostable bag. In one embodiment, theproduct is a combination of polybutylene succinate with polymers such asPP, PS and PC, and with plastics such as polylactic acid,polyhydroxyalkanoate, and poly-3-hydroxy butyrateco-valerate. In oneembodiment, the product is a combination of polybutylene succinate withfibers or fillers for applications such as automotive interiors,nonwovens, construction materials and consumer goods.

In some embodiments, provided herein is a culture medium comprisingbioderived succinate. In some embodiments, the bioderived succinate isproduced by culturing a non-naturally occurring microbial organismhaving a methanol metabolic pathway and succinate pathway, as providedherein. In certain embodiments, the bioderived succinate has acarbon-12, carbon-13 and carbon-14 isotope ratio that reflects anatmospheric carbon dioxide uptake source. In one embodiment, the culturemedium is separated from a non-naturally occurring microbial organismhaving a methanol metabolic pathway and succinate pathway.

In other embodiments, provided herein is a bioderived succinate. In someembodiments, the bioderived succinate is produced by culturing anon-naturally occurring microbial organism having a methanol metabolicpathway and succinate pathway, as provided herein. In certainembodiments, the bioderived succinate has a carbon-12, carbon-13 andcarbon-14 isotope ratio that reflects an atmospheric carbon dioxideuptake source. In some embodiments, the bioderived succinate has an Fmvalue of at least 80%, at least 85%, at least 90%, at least 95% or atleast 98%. In certain embodiments, the bioderived succinate is acomponent of culture medium.

In certain embodiments, provided herein is a composition comprising abioderived succinate provided herein, for example, a bioderivedsuccinate produced by culturing a non-naturally occurring microbialorganism having a methanol metabolic pathway and succinate pathway, asprovided herein. In some embodiments, the composition further comprisesa compound other than said bioderived succinate. In certain embodiments,the compound other than said bioderived succinate is a trace amount of acellular portion of a non-naturally occurring microbial organism havinga methanol metabolic pathway and a succinate pathway, as providedherein.

In some embodiments, provided herein is a biobased product comprising abioderived succinate provided herein. In certain embodiments, thebiobased product is a butanediol, tetrahydrofuran, pyrrolidone, solvent,paint, deicer, plastic, fuel additive, fabric, carpet, pigment,detergent, metal plating, polymer, polybutylene succinate polymer,biodegradable plastic, flexible packaging, agricultural film,compostable bag; a combination of polybutylene succinate with polymerssuch as PP, PS and PC, and with plastics such as polylactic acid,polyhydroxyalkanoate, and poly-3-hydroxy butyrateco-valerate; and acombination of polybutylene succinate with fibers or fillers forapplications such as automotive interiors, nonwovens, constructionmaterials and consumer goods, and the like. In certain embodiments, thebiobased product comprises at least 5% bioderived succinate. In certainembodiments, the biobased product comprises at least 10% bioderivedsuccinate. In some embodiments, the biobased product comprises at least20% bioderived succinate. In other embodiments, the biobased productcomprises at least 30% bioderived succinate. In some embodiments, thebiobased product comprises at least 40% bioderived succinate. In otherembodiments, the biobased product comprises at least 50% bioderivedsuccinate. In one embodiment, the biobased product comprises a portionof said bioderived succinate as a repeating unit. In another embodiment,provided herein is a molded product obtained by molding the biobasedproduct provided herein. In other embodiments, provided herein is aprocess for producing a biobased product provided herein, comprisingchemically reacting said bioderived succinate with itself or anothercompound in a reaction that produces said biobased product. In certainembodiments, provided herein is a polymer comprising or obtained byconverting the bioderived succinate. In other embodiments, providedherein is a method for producing a polymer, comprising chemically ofenzymatically converting the bioderived succinate to the polymer. In yetother embodiments, provided herein is a composition comprising thebioderived succinate, or a cell lysate or culture supernatant thereof.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing the referenced metabolic reaction,reactant or product. Unless otherwise expressly stated herein, thoseskilled in the art will understand that reference to a reaction alsoconstitutes reference to the reactants and products of the reaction.Similarly, unless otherwise expressly stated herein, reference to areactant or product also references the reaction and that reference toany of these metabolic constitutes also references the gene or genesencoding the enzymes that catalyze the referenced reaction, reactant orproduct. Likewise, given the well known fields of metabolicbiochemistry, enzymology and genomics, reference herein to a gene orencoding nucleic acid also constitutes a reference to the correspondingencoded enzyme and the reaction it catalyzes, or a protein associatedwith the reaction, as well as the reactants and products of thereaction.

Microbial organisms generally lack the capacity to synthesize succinate,and therefore any of the compounds disclosed herein to be within thesuccinate family of compounds, or otherwise known by those in the art tobe within the succinate family of compounds. Moreover, organisms havingall of the requisite metabolic enzymatic capabilities are not known toproduce succinate from the enzymes described and biochemical pathwaysexemplified herein. In contrast, the non-naturally occurring microbialorganisms of the invention can generate succinate as a product, as wellas intermediates thereof. The biosynthesis of succinate, as well asintermediates thereof, is particularly useful in chemical synthesis ofsuccinate family of compounds, it also allows for the furtherbiosynthesis of succinate family compounds and avoids altogetherchemical synthesis procedures.

The non-naturally occurring microbial organisms of the invention thatcan produce succinate are produced by ensuring that a host microbialorganism includes functional capabilities for the complete biochemicalsynthesis of at least one succinate biosynthetic pathway of theinvention. Ensuring at least one requisite succinate biosyntheticpathway confers succinate biosynthesis capability onto the hostmicrobial organism.

The organisms and methods are described herein with general reference tothe metabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art will understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reaction,and reference to any of these metabolic constituents also references thegene or genes encoding the enzymes that catalyze or proteins involved inthe referenced reaction, reactant or product. Likewise, given the wellknown fields of metabolic biochemistry, enzymology and genomics,reference herein to a gene or encoding nucleic acid also constitutes areference to the corresponding encoded enzyme and the reaction itcatalyzes or a protein associated with the reaction as well as thereactants and products of the reaction.

The non-naturally occurring microbial organisms described herein can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes or proteins participating in one or more methanolmetabolic, formaldehyde assimilation, and/or succinate biosyntheticpathways. Depending on the host microbial organism chosen forbiosynthesis, nucleic acids for some or all of a particular methanolmetabolic, formaldehyde assimilation, and/or succinate biosyntheticpathway can be expressed. For example, if a chosen host is deficient inone or more enzymes or proteins for a desired metabolic, assimilation,or biosynthetic pathway, then expressible nucleic acids for thedeficient enzyme(s) or protein(s) are introduced into the host forsubsequent exogenous expression. Alternatively, if the chosen hostexhibits endogenous expression of some pathway genes, but is deficientin others, then an encoding nucleic acid is needed for the deficientenzyme(s) or protein(s) to achieve succinate biosynthesis and/ormethanol metabolism. Thus, a non-naturally occurring microbial organismdescribed herein can be produced by introducing exogenous enzyme orprotein activities to obtain a desired metabolic pathway and/or adesired biosynthetic pathway can be obtained by introducing one or moreexogenous enzyme or protein activities that, together with one or moreendogenous enzymes or proteins, produces a desired product such assuccinate.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable tofermentation processes. Exemplary bacteria include species selected fromEscherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.Exemplary yeasts or fungi include species selected from Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichiapastoris, Rhizopus arrhizus, Rhizobus oryzae, and the like. E. coli is aparticularly useful host organism since it is a well characterizedmicrobial organism suitable for genetic engineering. Other particularlyuseful host organisms include yeast such as Saccharomyces cerevisiae. Itis understood that any suitable microbial host organism can be used tointroduce metabolic and/or genetic modifications to produce a desiredproduct.

Depending on the succinate biosynthetic, methanol metabolic and/orformaldehyde assimilation pathway constituents of a selected hostmicrobial organism, the non-naturally occurring microbial organismsprovided herein will, in some embodiments, include at least oneexogenously expressed succinate, formaldehyde assimilation and/ormethanol metabolic pathway-encoding nucleic acid and up to all encodingnucleic acids for one or more succinate biosynthetic pathways,formaldehyde assimilation pathways and/or methanol metabolic pathways.For example, succinate biosynthesis can be established in a hostdeficient in a pathway enzyme or protein through exogenous expression ofthe corresponding encoding nucleic acid. In a host deficient in allenzymes or proteins of a succinate pathway, exogenous expression of allenzyme or proteins in the pathway can be included, although it isunderstood that all enzymes or proteins of a pathway can be expressedeven if the host contains at least one of the pathway enzymes orproteins. For example, exogenous expression of all enzymes or proteinsin a pathway for production of succinate can be included. The same holdstrue for the methanol metabolic pathways and formaldehyde assimilationpathways provided herein. In some embodiments, the nucleic acid encodinga succinate pathway enzyme is an exogenous nucleic acid. In otherembodiments, the nucleic acid encoding an succinate pathway enzyme is anendogenous nucleic acid.

In certain embodiments, the non-naturally occurring microbial organismcomprises (1) a methanol metabolic pathway, wherein said organismcomprises one or more exogenous nucleic acids encoding a methanolmetabolic pathway enzyme provided herein, and (2) a succinate pathway,but the microbial organism does not further comprise one or moreexogenous nucleic acids encoding a succinate pathway enzyme providedherein. In some embodiments, succinate pathway enzyme(s) provided hereinare endogenous to the microbial organism and the nucleic acid encodingan succinate pathway enzyme is an endogenous nucleic acid. In otherembodiments, only a single succinate pathway enzyme is encoded by anexogenous nucleic acid, e.g., a malic enzyme (FIG. 2, step G), whereasthe remaining succinate pathway enzymes are encoded by one or moreendogenous nucleic acids.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel the succinatepathway, formaldehyde assimilation pathway, and methanol metabolicpathway deficiencies of the selected host microbial organism. Therefore,a non-naturally occurring microbial organism of the invention can haveone, two, three, four, five, six, seven, eight, nine, or up to allnucleic acids encoding the enzymes or proteins constituting a methanolmetabolic pathway, formaldehyde assimilation pathway, and/or succinatebiosynthetic pathway disclosed herein. In some embodiments, thenon-naturally occurring microbial organisms also can include othergenetic modifications that facilitate or optimize succinatebiosynthesis, formaldehyde assimilation, and/or methanol metabolism orthat confer other useful functions onto the host microbial organism. Onesuch other functionality can include, for example, augmentation of thesynthesis of one or more of the succinate pathway precursors.

Generally, a host microbial organism is selected such that it producesthe precursor of a succinate pathway, either as a naturally producedmolecule or as an engineered product that either provides de novoproduction of a desired precursor or increased production of a precursornaturally produced by the host microbial organism. A host organism canbe engineered to increase production of a precursor, as disclosedherein. In addition, a microbial organism that has been engineered toproduce a desired precursor can be used as a host organism and furtherengineered to express enzymes or proteins of a succinate pathway.

In some embodiments, a non-naturally occurring microbial organismprovided herein is generated from a host that contains the enzymaticcapability to synthesize succinate, assimilate formaldehyde and/ormetabolize methanol. In this specific embodiment it can be useful toincrease the synthesis or accumulation of a succinate pathway product,formaldehyde assimilation pathway product and/or methanol metabolicpathway product (e.g., reducing equivalents and/or formaldehyde) to, forexample, drive succinate pathway reactions toward succinate production.Increased synthesis or accumulation can be accomplished by, for example,overexpression of nucleic acids encoding one or more of theabove-described succinate, formaldehyde assimilation and/or methanolmetabolic pathway enzymes or proteins. Over expression the enzyme(s)and/or protein(s) of the succinate pathway, formaldehyde assimilation,and/or methanol metabolic pathway can occur, for example, throughexogenous expression of the endogenous gene(s), or through exogenousexpression of the heterologous gene(s). Therefore, naturally occurringorganisms can be readily generated to be non-naturally occurringmicrobial organisms, for example, producing succinate throughoverexpression of one, two, three, four, five, six, seven, eight, up toall nucleic acids encoding succinate biosynthetic pathway, and/ormethanol metabolic pathway enzymes or proteins. Naturally occurringorganisms can also be readily generated to be non-naturally occurringmicrobial organisms, for example, assimilating formaldehyde, throughoverexpression of one, two, three, four, five, six, seven, eight, up toall nucleic acids encoding formaldehyde assimilation pathway, and/ormethanol metabolic pathway enzymes or proteins. In addition, anon-naturally occurring organism can be generated by mutagenesis of anendogenous gene that results in an increase in activity of an enzyme inthe succinate, formaldehyde assimilation and/or methanol metabolicpathway biosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

It is understood that, in methods provided herein, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism provided herein.The nucleic acids can be introduced so as to confer, for example, asuccinate biosynthetic, formaldehyde assimilation and/or methanolmetabolic pathway onto the microbial organism. Alternatively, encodingnucleic acids can be introduced to produce an intermediate microbialorganism having the biosynthetic capability to catalyze some of therequired reactions to confer succinate biosynthetic, formaldehydeassimilation and/or methanol metabolic capability. For example, anon-naturally occurring microbial organism having a succinatebiosynthetic pathway, formaldehyde assimilation pathway and/or methanolmetabolic pathway can comprise at least two exogenous nucleic acidsencoding desired enzymes or proteins. Thus, it is understood that anycombination of two or more enzymes or proteins of a biosyntheticpathway, formaldehyde assimilation pathway and/or metabolic pathway canbe included in a non-naturally occurring microbial organism providedherein. Similarly, it is understood that any combination of three ormore enzymes or proteins of a biosynthetic pathway, formaldehydeassimilation pathway and/or metabolic pathway can be included in anon-naturally occurring microbial organism of the invention, as desired,so long as the combination of enzymes and/or proteins of the desiredbiosynthetic pathway, formaldehyde assimilation pathway and/or metabolicpathway results in production of the corresponding desired product.Similarly, any combination of four or more enzymes or proteins of abiosynthetic pathway, formaldehyde assimilation pathway and/or methanolmetabolic pathway as disclosed herein can be included in a non-naturallyoccurring microbial organism provided herein, as desired, so long as thecombination of enzymes and/or proteins of the desired biosynthetic,assimilation and/or metabolic pathway results in production of thecorresponding desired product.

In addition to the metabolism of methanol, assimilation of formaldehyde,and biosynthesis of succinate, as described herein, the non-naturallyoccurring microbial organisms and methods provided also can be utilizedin various combinations with each other and with other microbialorganisms and methods well known in the art to achieve productbiosynthesis by other routes. For example, one alternative to producesuccinate, other than use of the succinate producers is through additionof another microbial organism capable of converting a succinate pathwayintermediate to succinate. One such procedure includes, for example, thefermentation of a microbial organism that produces a succinate pathwayintermediate. The succinate pathway intermediate can then be used as asubstrate for a second microbial organism that converts the succinatepathway intermediate to succinate. The succinate pathway intermediatecan be added directly to another culture of the second organism or theoriginal culture of the succinate pathway intermediate producers can bedepleted of these microbial organisms by, for example, cell separation,and then subsequent addition of the second organism to the fermentationbroth can be utilized to produce the final product without intermediatepurification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, succinate. In theseembodiments, biosynthetic pathways for a desired product can besegregated into different microbial organisms, and the differentmicrobial organisms can be co-cultured to produce the final product. Insuch a biosynthetic scheme, the product of one microbial organism is thesubstrate for a second microbial organism until the final product issynthesized. For example, the biosynthesis of succinate can beaccomplished by constructing a microbial organism that containsbiosynthetic pathways for conversion of one pathway intermediate toanother pathway intermediate or the product. Alternatively, succinatealso can be biosynthetically produced from microbial organisms throughco-culture or co-fermentation using two organisms in the same vessel,where the first microbial organism produces a succinate intermediate andthe second microbial organism converts the intermediate to succinate.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methodstogether with other microbial organisms, with the co-culture of othernon-naturally occurring microbial organisms having subpathways and withcombinations of other chemical and/or biochemical procedures well knownin the art to produce succinate and/or metabolize methanol.

Sources of encoding nucleic acids for a succinate, formaldehydeassimilation, or methanol metabolic pathway enzyme or protein caninclude, for example, any species where the encoded gene product iscapable of catalyzing the referenced reaction. Such species include bothprokaryotic and eukaryotic organisms including, but not limited to,bacteria, including archaea and eubacteria, and eukaryotes, includingyeast, plant, insect, animal, and mammal, including human. Exemplaryspecies for such sources include, for example, Escherichia coli,Saccharomyces cerevisiae, Saccharomyces kluyveri, Candida boidinii,Clostridium kluyveri, Clostridium acetobutylicum, Clostridiumbeijerinckii, Clostridium saccharoperbutylacetonicum, Clostridiumperfringens, Clostridium difficile, Clostridium botulinum, Clostridiumtyrobutyricum, Clostridium tetanomorphum, Clostridium tetani,Clostridium propionicum, Clostridium aminobutyricum, Clostridiumsubterminale, Clostridium sticklandii, Ralstonia eutropha, Mycobacteriumbovis, Mycobacterium tuberculosis, Porphyromonas gingivalis, Arabidopsisthaliana, Thermus thermophilus, Pseudomonas species, includingPseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri,Pseudomonas fluorescens, Homo sapiens, Oryctolagus cuniculus,Rhodobacter spaeroides, Thermoanaerobacter brockii, Metallosphaerasedula, Leuconostoc mesenteroides, Chloroflexus aurantiacus, Roseiflexuscastenholzii, Erythrobacter, Simmondsia chinensis, Acinetobacterspecies, including Acinetobacter calcoaceticus and Acinetobacter baylyi,Porphyromonas gingivalis, Sulfolobus tokodaii, Sulfolobus solfataricus,Sulfolobus acidocaldarius, Bacillus subtilis, Bacillus cereus, Bacillusmegaterium, Bacillus brevis, Bacillus pumilus, Rattus norvegicus,Klebsiella pneumonia, Klebsiella oxytoca, Euglena gracilis, Treponemadenticola, Moorella thermoacetica, Thermotoga maritima, Halobacteriumsalinarum, Geobacillus stearothermophilus, Aeropyrum pernix, Sus scrofa,Caenorhabditis elegans, Corynebacterium glutamicum, Acidaminococcusfermentans, Lactococcus lactis, Lactobacillus plantarum, Streptococcusthermophilus, Enterobacter aerogenes, Candida, Aspergillus terreus,Pedicoccus pentosaceus, Zymomonas mobilus, Acetobacter pasteurians,Kluyveromyces lactis, Eubacterium barkeri, Bacteroides capillosus,Anaerotruncus colihominis, Natranaerobius thermophilusm, Campylobacterjejuni, Haemophilus influenzae, Serratia marcescens, Citrobacteramalonaticus, Myxococcus xanthus, Fusobacterium nuleatum, Penicilliumchrysogenum, marine gamma proteobacterium, butyrate-producing bacterium,Nocardia iowensis, Nocardia farcinica, Streptomyces griseus,Schizosaccharomyces pombe, Geobacillus thermoglucosidasius, Salmonellatyphimurium, Vibrio cholera, Heliobacter pylori, Nicotiana tabacum,Oryza sativa, Haloferax mediterranei, Agrobacterium tumefaciens,Achromobacter denitrificans, Fusobacterium nucleatum, Streptomycesclavuligenus, Acinetobacter baumanii, Mus musculus, Lachancea kluyveri,Trichomonas vaginalis, Trypanosoma brucei, Pseudomonas stutzeri,Bradyrhizobium japonicum, Mesorhizobium loti, Bos taurus, Nicotianaglutinosa, Vibrio vulnificus, Selenomonas ruminantium, Vibrioparahaemolyticus, Archaeoglobus fulgidus, Haloarcula marismortui,Pyrobaculum aerophilum, Mycobacterium smegmatis MC2 155, Mycobacteriumavium subsp. paratuberculosis K-10, Mycobacterium marinum M,Tsukamurella paurometabola DSM 20162, Cyanobium PCC7001, Dictyosteliumdiscoideum AX4, as well as other exemplary species disclosed herein oravailable as source organisms for corresponding genes. However, with thecomplete genome sequence available for now more than 550 species (withmore than half of these available on public databases such as the NCBI),including 395 microorganism genomes and a variety of yeast, fungi,plant, and mammalian genomes, the identification of genes encoding therequisite succinate biosynthetic activity for one or more genes inrelated or distant species, including for example, homologues,orthologs, paralogs and nonorthologous gene displacements of knowngenes, and the interchange of genetic alterations between organisms isroutine and well known in the art. Accordingly, the metabolicalterations allowing biosynthesis of succinate, metabolism of methanoland/or assimilation of formaldehyde described herein with reference to aparticular organism such as E. coli can be readily applied to othermicroorganisms, including prokaryotic and eukaryotic organisms alike.Given the teachings and guidance provided herein, those skilled in theart will know that a metabolic alteration exemplified in one organismcan be applied equally to other organisms.

In some instances, such as when an alternative succinate biosynthetic,formaldehyde assimilation and/or methanol metabolic pathway exists in anunrelated species, succinate biosynthesis, formaldehyde assimilationand/or methanol metabolism can be conferred onto the host species by,for example, exogenous expression of a paralog or paralogs from theunrelated species that catalyzes a similar, yet non-identical metabolicreaction to replace the referenced reaction. Because certain differencesamong metabolic networks exist between different organisms, thoseskilled in the art will understand that the actual gene usage betweendifferent organisms may differ. However, given the teachings andguidance provided herein, those skilled in the art also will understandthat the teachings and methods provided herein can be applied to allmicrobial organisms using the cognate metabolic alterations to thoseexemplified herein to construct a microbial organism in a species ofinterest that will synthesize succinate, assimilate formaldehyde, and/ormetabolize methanol.

Methods for constructing and testing the expression levels of anon-naturally occurring succinate-producing host can be performed, forexample, by recombinant and detection methods well known in the art.Such methods can be found described in, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring HarborLaboratory, New York (2001); and Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for metabolism ofmethanol, assimilation of formaldehyde and/or production of succinatecan be introduced stably or transiently into a host cell usingtechniques well known in the art including, but not limited to,conjugation, electroporation, chemical transformation, transduction,transfection, and ultrasound transformation. For exogenous expression inE. coli or other prokaryotic cells, some nucleic acid sequences in thegenes or cDNAs of eukaryotic nucleic acids can encode targeting signalssuch as an N-terminal mitochondrial or other targeting signal, which canbe removed before transformation into prokaryotic host cells, ifdesired. For example, removal of a mitochondrial leader sequence led toincreased expression in E. coli (Hoffmeister et al., J. Biol. Chem.280:4329-4338 (2005)). For exogenous expression in yeast or othereukaryotic cells, genes can be expressed in the cytosol without theaddition of leader sequence, or can be targeted to mitochondrion orother organelles, or targeted for secretion, by the addition of asuitable targeting sequence such as a mitochondrial targeting orsecretion signal suitable for the host cells. Thus, it is understoodthat appropriate modifications to a nucleic acid sequence to remove orinclude a targeting sequence can be incorporated into an exogenousnucleic acid sequence to impart desirable properties. Furthermore, genescan be subjected to codon optimization with techniques well known in theart to achieve optimized expression of the proteins.

An expression vector or vectors can be constructed to include one ormore succinate biosynthetic, formaldehyde assimilation and/or methanolmetabolic pathway encoding nucleic acids as exemplified herein operablylinked to expression control sequences functional in the host organism.Expression vectors applicable for use in the microbial host organismsprovided include, for example, plasmids, phage vectors, viral vectors,episomes and artificial chromosomes, including vectors and selectionsequences or markers operable for stable integration into a hostchromosome. Additionally, the expression vectors can include one or moreselectable marker genes and appropriate expression control sequences.Selectable marker genes also can be included that, for example, provideresistance to antibiotics or toxins, complement auxotrophicdeficiencies, or supply critical nutrients not in the culture media.Expression control sequences can include constitutive and induciblepromoters, transcription enhancers, transcription terminators, and thelike which are well known in the art. When two or more exogenousencoding nucleic acids are to be co-expressed, both nucleic acids can beinserted, for example, into a single expression vector or in separateexpression vectors. For single vector expression, the encoding nucleicacids can be operationally linked to one common expression controlsequence or linked to different expression control sequences, such asone inducible promoter and one constitutive promoter. The transformationof exogenous nucleic acid sequences involved in a metabolic or syntheticpathway can be confirmed using methods well known in the art. Suchmethods include, for example, nucleic acid analysis such as Northernblots or polymerase chain reaction (PCR) amplification of mRNA, orimmunoblotting for expression of gene products, or other suitableanalytical methods to test the expression of an introduced nucleic acidsequence or its corresponding gene product. It is understood by thoseskilled in the art that the exogenous nucleic acid is expressed in asufficient amount to produce the desired product, and it is furtherunderstood that expression levels can be optimized to obtain sufficientexpression using methods well known in the art and as disclosed herein.

Suitable purification and/or assays to test, e.g., for the production ofsuccinate can be performed using well known methods. Suitable replicatessuch as triplicate cultures can be grown for each engineered strain tobe tested. For example, product and byproduct formation in theengineered production host can be monitored. The final product andintermediates, and other organic compounds, can be analyzed by methodssuch as HPLC (High Performance Liquid Chromatography), GC-MS (GasChromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-MassSpectroscopy) or other suitable analytical methods using routineprocedures well known in the art. The release of product in thefermentation broth can also be tested with the culture supernatant.Byproducts and residual glucose can be quantified by HPLC using, forexample, a refractive index detector for glucose and alcohols, and a UVdetector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779(2005)), or other suitable assay and detection methods well known in theart. The individual enzyme or protein activities from the exogenous DNAsequences can also be assayed using methods well known in the art.

The succinate can be separated from other components in the cultureusing a variety of methods well known in the art. Such separationmethods include, for example, extraction procedures as well as methodsthat include continuous liquid-liquid extraction, pervaporation,membrane filtration, membrane separation, reverse osmosis,electrodialysis, distillation, crystallization, centrifugation,extractive filtration, ion exchange chromatography, size exclusionchromatography, adsorption chromatography, and ultrafiltration. All ofthe above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products, orintermediates thereof. For example, the succinate producers can becultured for the biosynthetic production of succinate. Accordingly, insome embodiments, the invention provides culture medium having asuccinate, formaldehyde assimilation and/or methanol metabolic pathwayintermediate described herein. In some aspects, the culture medium canalso be separated from the non-naturally occurring microbial organismsprovided herein that produced the succinate, formaldehyde assimilationand/or methanol metabolic pathway intermediate. Methods for separating amicrobial organism from culture medium are well known in the art.Exemplary methods include filtration, flocculation, precipitation,centrifugation, sedimentation, and the like.

In certain embodiments, for example, for the production of theproduction of succinate, the recombinant strains are cultured in amedium with carbon source and other essential nutrients. It is sometimesdesirable and can be highly desirable to maintain anaerobic conditionsin the fermenter to reduce the cost of the overall process. Suchconditions can be obtained, for example, by first sparging the mediumwith nitrogen and then sealing the flasks with a septum and crimp-cap.For strains where growth is not observed anaerobically, microaerobic orsubstantially anaerobic conditions can be applied by perforating theseptum with a small hole for limited aeration. Exemplary anaerobicconditions have been described previously and are well-known in the art.Exemplary aerobic and anaerobic conditions are described, for example,in U.S. Publ. No. 2009/0047719. Fermentations can be performed in abatch, fed-batch or continuous manner, as disclosed herein.Fermentations can be performed in a batch, fed-batch or continuousmanner, as disclosed herein. Fermentations can also be conducted in twophases, if desired. The first phase can be aerobic to allow for highgrowth and therefore high productivity, followed by an anaerobic phaseof high succinate yields.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

The growth medium, can include, for example, any carbohydrate sourcewhich can supply a source of carbon to the non-naturally occurringmicroorganism. Such sources include, for example, sugars such asglucose, xylose, arabinose, galactose, mannose, fructose, sucrose andstarch; or glycerol, alone as the sole source of carbon or incombination with other carbon sources described herein or known in theart. In one embodiment, the carbon source is a sugar. In one embodiment,the carbon source is a sugar-containing biomass. In some embodiments,the sugar is glucose. In one embodiment, the sugar is xylose. In anotherembodiment, the sugar is arabinose. In one embodiment, the sugar isgalactose. In another embodiment, the sugar is fructose. In otherembodiments, the sugar is sucrose. In one embodiment, the sugar isstarch. In certain embodiments, the carbon source is glycerol. In someembodiments, the carbon source is crude glycerol. In one embodiment, thecarbon source is crude glycerol without treatment. In other embodiments,the carbon source is glycerol and glucose. In another embodiment, thecarbon source is methanol and glycerol. In one embodiment, the carbonsource is carbon dioxide. In one embodiment, the carbon source isformate. In one embodiment, the carbon source is methane. In oneembodiment, the carbon source is methanol. In one embodiment, the carbonsource is chemoelectro-generated carbon (see, e.g., Liao et al. (2012)Science 335:1596). In one embodiment, the chemoelectro-generated carbonis methanol. In one embodiment, the chemoelectro-generated carbon isformate. In one embodiment, the chemoelectro-generated carbon is formateand methanol. In one embodiment, the carbon source is a sugar andmethanol. In another embodiment, the carbon source is a sugar andglycerol. In other embodiments, the carbon source is a sugar and crudeglycerol. In yet other embodiments, the carbon source is a sugar andcrude glycerol without treatment. In one embodiment, the carbon sourceis a sugar-containing biomass and methanol. In another embodiment, thecarbon source is a sugar-containing biomass and glycerol. In otherembodiments, the carbon source is a sugar-containing biomass and crudeglycerol. In yet other embodiments, the carbon source is asugar-containing biomass and crude glycerol without treatment. Othersources of carbohydrate include, for example, renewable feedstocks andbiomass. Exemplary types of biomasses that can be used as feedstocks inthe methods of the invention include cellulosic biomass, hemicellulosicbiomass and lignin feedstocks or portions of feedstocks. Such biomassfeedstocks contain, for example, carbohydrate substrates useful ascarbon sources such as glucose, xylose, arabinose, galactose, mannose,fructose and starch. Given the teachings and guidance provided herein,those skilled in the art will understand that renewable feedstocks andbiomass other than those exemplified above also can be used forculturing the microbial organisms provided herein for the production ofsuccinate and other pathway intermediates.

In one embodiment, the carbon source is glycerol. In certainembodiments, the glycerol carbon source is crude glycerol or crudeglycerol without further treatment. In a further embodiment, the carbonsource comprises glycerol or crude glycerol, and also sugar or asugar-containing biomass, such as glucose. In a specific embodiment, theconcentration of glycerol in the fermentation broth is maintained byfeeding crude glycerol, or a mixture of crude glycerol and sugar (e.g.,glucose). In certain embodiments, sugar is provided for sufficientstrain growth. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of glycerol to sugar of from200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of glycerol to sugar of from100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of glycerol to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of glycerol to sugar of from 50:1 to 5:1.In certain embodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 100:1. In one embodiment,the sugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 90:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 80:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 70:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 60:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 50:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 40:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 30:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 20:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 10:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 5:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 2:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:1. In certain embodiments,the sugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 1:100. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 1:90.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:80. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 1:70. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 1:60.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:50. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 1:40. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 1:30.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:20. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 1:10. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 1:5.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:2. In certain embodimentsof the ratios provided above, the sugar is a sugar-containing biomass.In certain other embodiments of the ratios provided above, the glycerolis a crude glycerol or a crude glycerol without further treatment. Inother embodiments of the ratios provided above, the sugar is asugar-containing biomass, and the glycerol is a crude glycerol or acrude glycerol without further treatment.

Crude glycerol can be a by-product produced in the production ofbiodiesel, and can be used for fermentation without any furthertreatment. Biodiesel production methods include (1) a chemical methodwherein the glycerol-group of vegetable oils or animal oils issubstituted by low-carbon alcohols such as methanol or ethanol toproduce a corresponding fatty acid methyl esters or fatty acid ethylesters by transesterification in the presence of acidic or basiccatalysts; (2) a biological method where biological enzymes or cells areused to catalyze transesterification reaction and the correspondingfatty acid methyl esters or fatty acid ethyl esters are produced; and(3) a supercritical method, wherein transesterification reaction iscarried out in a supercritical solvent system without any catalysts. Thechemical composition of crude glycerol can vary with the process used toproduce biodiesel, the transesterification efficiency, recoveryefficiency of the biodiesel, other impurities in the feedstock, andwhether methanol and catalysts were recovered. For example, the chemicalcompositions of eleven crude glycerol collected from seven Australianbiodiesel producers reported that glycerol content ranged between 38%and 96%, with some samples including more than 14% methanol and 29% ash.In certain embodiments, the crude glycerol comprises from 5% to 99%glycerol. In some embodiments, the crude glycerol comprises from 10% to90% glycerol. In some embodiments, the crude glycerol comprises from 10%to 80% glycerol. In some embodiments, the crude glycerol comprises from10% to 70% glycerol. In some embodiments, the crude glycerol comprisesfrom 10% to 60% glycerol. In some embodiments, the crude glycerolcomprises from 10% to 50% glycerol. In some embodiments, the crudeglycerol comprises from 10% to 40% glycerol. In some embodiments, thecrude glycerol comprises from 10% to 30% glycerol. In some embodiments,the crude glycerol comprises from 10% to 20% glycerol. In someembodiments, the crude glycerol comprises from 80% to 90% glycerol. Insome embodiments, the crude glycerol comprises from 70% to 90% glycerol.In some embodiments, the crude glycerol comprises from 60% to 90%glycerol. In some embodiments, the crude glycerol comprises from 50% to90% glycerol. In some embodiments, the crude glycerol comprises from 40%to 90% glycerol. In some embodiments, the crude glycerol comprises from30% to 90% glycerol. In some embodiments, the crude glycerol comprisesfrom 20% to 90% glycerol. In some embodiments, the crude glycerolcomprises from 20% to 40% glycerol. In some embodiments, the crudeglycerol comprises from 40% to 60% glycerol. In some embodiments, thecrude glycerol comprises from 60% to 80% glycerol. In some embodiments,the crude glycerol comprises from 50% to 70% glycerol. In oneembodiment, the glycerol comprises 5% glycerol. In one embodiment, theglycerol comprises 10% glycerol. In one embodiment, the glycerolcomprises 15% glycerol. In one embodiment, the glycerol comprises 20%glycerol. In one embodiment, the glycerol comprises 25% glycerol. In oneembodiment, the glycerol comprises 30% glycerol. In one embodiment, theglycerol comprises 35% glycerol. In one embodiment, the glycerolcomprises 40% glycerol. In one embodiment, the glycerol comprises 45%glycerol. In one embodiment, the glycerol comprises 50% glycerol. In oneembodiment, the glycerol comprises 55% glycerol. In one embodiment, theglycerol comprises 60% glycerol. In one embodiment, the glycerolcomprises 65% glycerol. In one embodiment, the glycerol comprises 70%glycerol. In one embodiment, the glycerol comprises 75% glycerol. In oneembodiment, the glycerol comprises 80% glycerol. In one embodiment, theglycerol comprises 85% glycerol. In one embodiment, the glycerolcomprises 90% glycerol. In one embodiment, the glycerol comprises 95%glycerol. In one embodiment, the glycerol comprises 99% glycerol.

In one embodiment, the carbon source is methanol or formate. In certainembodiments, methanol is used as a carbon source in the formaldehydeassimilation pathways provided herein. In one embodiment, the carbonsource is methanol or formate. In other embodiments, formate is used asa carbon source in the formaldehyde assimilation pathways providedherein. In specific embodiments, methanol is used as a carbon source inthe methanol metabolic pathways provided herein, either alone or incombination with the product pathways provided herein.

In one embodiment, the carbon source comprises methanol, and sugar(e.g., glucose) or a sugar-containing biomass. In another embodiment,the carbon source comprises formate, and sugar (e.g., glucose) or asugar-containing biomass. In one embodiment, the carbon source comprisesmethanol, formate, and sugar (e.g., glucose) or a sugar-containingbiomass. In specific embodiments, the methanol or formate, or both, inthe fermentation feed is provided as a mixture with sugar (e.g.,glucose) or sugar-comprising biomass. In certain embodiments, sugar isprovided for sufficient strain growth.

In certain embodiments, the carbon source comprises methanol and a sugar(e.g., glucose). In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol to sugar of from200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol to sugar of from100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of methanol to sugar of from 50:1 to 5:1.In certain embodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 100:1. In one embodiment,the sugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 90:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 80:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 70:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 60:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 50:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 40:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 30:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 20:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 10:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 5:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 2:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:1. In certain embodiments,the sugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:100. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:90.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:80. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:70. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:60.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:50. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:40. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:30.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:20. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:10. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:5.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:2. In certain embodimentsof the ratios provided above, the sugar is a sugar-containing biomass.

In certain embodiments, the carbon source comprises formate and a sugar(e.g., glucose). In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of from200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of from100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of formate to sugar of from 50:1 to 5:1.In certain embodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 100:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 90:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 80:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 70:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 60:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 50:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 40:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 30:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 20:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 10:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 5:1. In one embodiment, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of 2:1. Inone embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:1. In certain embodiments,the sugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:100. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:90.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:80. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:70. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:60.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:50. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:40. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:30.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:20. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:10. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:5.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:2. In certain embodimentsof the ratios provided above, the sugar is a sugar-containing biomass.

In certain embodiments, the carbon source comprises a mixture ofmethanol and formate, and a sugar (e.g., glucose). In certainembodiments, sugar is provided for sufficient strain growth. In someembodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of from 200:1 to1:200. In some embodiments, the sugar (e.g., glucose) is provided at amolar concentration ratio of methanol and formate to sugar of from 100:1to 1:100. In some embodiments, the sugar (e.g., glucose) is provided ata molar concentration ratio of methanol and formate to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of methanol and formate to sugar of from50:1 to 5:1. In certain embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol and formate to sugarof 100:1. In one embodiment, the sugar (e.g., glucose) is provided at amolar concentration ratio of methanol and formate to sugar of 90:1. Inone embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 80:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 70:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 60:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 50:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 40:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 30:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 20:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 10:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 5:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 2:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:1. In certainembodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:100. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:90. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:80. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:70. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:60. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:50. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:40. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:30. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:20. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:10. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:5. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:2. In certainembodiments of the ratios provided above, the sugar is asugar-containing biomass.

Given the teachings and guidance provided herein, those skilled in theart will understand that a non-naturally occurring microbial organismcan be produced that secretes the biosynthesized compounds when grown ona carbon source such as a carbohydrate. Such compounds include, forexample, succinate and any of the intermediate metabolites in thesuccinate pathway. All that is required is to engineer in one or more ofthe required enzyme or protein activities to achieve biosynthesis of thedesired compound or intermediate including, for example, inclusion ofsome or all of the succinate biosynthetic pathways. Accordingly,provided herein is a non-naturally occurring microbial organism thatproduces and/or secretes succinate when grown on a carbohydrate or othercarbon source and produces and/or secretes any of the intermediatemetabolites shown in the succinate pathway when grown on a carbohydrateor other carbon source. The succinate-producing microbial organismsprovided herein can initiate synthesis from an intermediate. The sameholds true for intermediates in the formaldehyde assimilation andmethanol metabolic pathways.

The non-naturally occurring microbial organisms provided herein areconstructed using methods well known in the art as exemplified herein toendogenously or exogenously express at least one nucleic acid encoding asuccinate biosynthetic pathway and/or exogenously express a methanolmetabolic pathway enzyme or protein in sufficient amounts to producesuccinate. It is understood that the microbial organisms are culturedunder conditions sufficient to produce succinate. Following theteachings and guidance provided herein, the non-naturally occurringmicrobial organisms can achieve biosynthesis of succinate, resulting inintracellular concentrations between about 0.1-500 mM or more.Generally, the intracellular concentration of succinate is between about3-150 mM, particularly between about 5-125 mM and more particularlybetween about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, ormore. Intracellular concentrations between and above each of theseexemplary ranges also can be achieved from the non-naturally occurringmicrobial organisms provided herein.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. Publ. No.2009/0047719. Any of these conditions can be employed with thenon-naturally occurring microbial organisms as well as other anaerobicconditions well known in the art. Under such anaerobic or substantiallyanaerobic conditions, the succinate producers can synthesize succinateat intracellular concentrations of 5-100 mM or more as well as all otherconcentrations exemplified herein. It is understood that, even thoughthe above description refers to intracellular concentrations, succinatecan produce succinate intracellularly and/or secrete the product intothe culture medium.

Exemplary fermentation processes include, but are not limited to,fed-batch fermentation and batch separation; fed-batch fermentation andcontinuous separation; and continuous fermentation and continuousseparation. In an exemplary batch fermentation protocol, the productionorganism is grown in a suitably sized bioreactor sparged with anappropriate gas. Under anaerobic conditions, the culture is sparged withan inert gas or combination of gases, for example, nitrogen, N2/CO2mixture, argon, helium, and the like. As the cells grow and utilize thecarbon source, additional carbon source(s) and/or other nutrients arefed into the bioreactor at a rate approximately balancing consumption ofthe carbon source and/or nutrients. The temperature of the bioreactor ismaintained at a desired temperature, generally in the range of 22-37degrees C., but the temperature can be maintained at a higher or lowertemperature depending on the growth characteristics of the productionorganism and/or desired conditions for the fermentation process. Growthcontinues for a desired period of time to achieve desiredcharacteristics of the culture in the fermenter, for example, celldensity, product concentration, and the like. In a batch fermentationprocess, the time period for the fermentation is generally in the rangeof several hours to several days, for example, 8 to 24 hours, or 1, 2,3, 4 or 5 days, or up to a week, depending on the desired cultureconditions. The pH can be controlled or not, as desired, in which case aculture in which pH is not controlled will typically decrease to pH 3-6by the end of the run. Upon completion of the cultivation period, thefermenter contents can be passed through a cell separation unit, forexample, a centrifuge, filtration unit, and the like, to remove cellsand cell debris. In the case where the desired product is expressedintracellularly, the cells can be lysed or disrupted enzymatically orchemically prior to or after separation of cells from the fermentationbroth, as desired, in order to release additional product. Thefermentation broth can be transferred to a product separations unit.Isolation of product occurs by standard separations procedures employedin the art to separate a desired product from dilute aqueous solutions.Such methods include, but are not limited to, liquid-liquid extractionusing a water immiscible organic solvent (e.g., toluene or othersuitable solvents, including but not limited to diethyl ether, ethylacetate, tetrahydrofuran (THF), methylene chloride, chloroform, benzene,pentane, hexane, heptane, petroleum ether, methyl tertiary butyl ether(MTBE), dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), andthe like) to provide an organic solution of the product, if appropriate,standard distillation methods, and the like, depending on the chemicalcharacteristics of the product of the fermenation process.

In an exemplary fully continuous fermentation protocol, the productionorganism is generally first grown up in batch mode in order to achieve adesired cell density. When the carbon source and/or other nutrients areexhausted, feed medium of the same composition is supplied continuouslyat a desired rate, and fermentation liquid is withdrawn at the samerate. Under such conditions, the product concentration in the bioreactorgenerally remains constant, as well as the cell density. The temperatureof the fermenter is maintained at a desired temperature, as discussedabove. During the continuous fermentation phase, it is generallydesirable to maintain a suitable pH range for optimized production. ThepH can be monitored and maintained using routine methods, including theaddition of suitable acids or bases to maintain a desired pH range. Thebioreactor is operated continuously for extended periods of time,generally at least one week to several weeks and up to one month, orlonger, as appropriate and desired. The fermentation liquid and/orculture is monitored periodically, including sampling up to every day,as desired, to assure consistency of product concentration and/or celldensity. In continuous mode, fermenter contents are constantly removedas new feed medium is supplied. The exit stream, containing cells,medium, and product, are generally subjected to a continuous productseparations procedure, with or without removing cells and cell debris,as desired. Continuous separations methods employed in the art can beused to separate the product from dilute aqueous solutions, includingbut not limited to continuous liquid-liquid extraction using a waterimmiscible organic solvent (e.g., toluene or other suitable solvents,including but not limited to diethyl ether, ethyl acetate,tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane,hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE),dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and thelike), standard continuous distillation methods, and the like, or othermethods well known in the art.

In addition to the culturing and fermentation conditions disclosedherein, growth condition for achieving biosynthesis of succinate caninclude the addition of an osmoprotectant to the culturing conditions.In certain embodiments, the non-naturally occurring microbial organismsprovided herein can be sustained, cultured or fermented as describedherein in the presence of an osmoprotectant. Briefly, an osmoprotectantrefers to a compound that acts as an osmolyte and helps a microbialorganism as described herein survive osmotic stress. Osmoprotectantsinclude, but are not limited to, betaines, amino acids, and the sugartrehalose. Non-limiting examples of such are glycine betaine, pralinebetaine, dimethylthetin, dimethylslfonioproprionate,3-dimethylsulfonio-2-methylproprionate, pipecolic acid,dimethylsulfonioacetate, choline, L-carnitine and ectoine. In oneaspect, the osmoprotectant is glycine betaine. It is understood to oneof ordinary skill in the art that the amount and type of osmoprotectantsuitable for protecting a microbial organism described herein fromosmotic stress will depend on the microbial organism used. The amount ofosmoprotectant in the culturing conditions can be, for example, no morethan about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM,no more than about 1.5 mM, no more than about 2.0 mM, no more than about2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no morethan about 7.0 mM, no more than about 10 mM, no more than about 50 mM,no more than about 100 mM or no more than about 500 mM.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of succinate, as well as other pathway intermediates,includes anaerobic culture or fermentation conditions. In certainembodiments, the non-naturally occurring microbial organisms providedcan be sustained, cultured or fermented under anaerobic or substantiallyanaerobic conditions. Briefly, anaerobic conditions refer to anenvironment devoid of oxygen. Substantially anaerobic conditionsinclude, for example, a culture, batch fermentation or continuousfermentation such that the dissolved oxygen concentration in the mediumremains between 0 and 10% of saturation. Substantially anaerobicconditions also includes growing or resting cells in liquid medium or onsolid agar inside a sealed chamber maintained with an atmosphere of lessthan 1% oxygen. The percent of oxygen can be maintained by, for example,sparging the culture with an N₂/CO₂ mixture or other suitable non-oxygengas or gases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of succinate. Exemplary growth proceduresinclude, for example, fed-batch fermentation and batch separation;fed-batch fermentation and continuous separation, or continuousfermentation and continuous separation. All of these processes are wellknown in the art. Fermentation procedures are particularly useful forthe biosynthetic production of commercial quantities of succinate.Generally, and as with non-continuous culture procedures, the continuousand/or near-continuous production of succinate will include culturing anon-naturally occurring succinate producing organism of the invention insufficient nutrients and medium to sustain and/or nearly sustain growthin an exponential phase. Continuous culture under such conditions can beincluded, for example, growth for 1 day, 2, 3, 4, 5, 6 or 7 days ormore. Additionally, continuous culture can include longer time periodsof 1 week, 2, 3, 4 or 5 or more weeks and up to several months.Alternatively, organisms provided can be cultured for hours, if suitablefor a particular application. It is to be understood that the continuousand/or near-continuous culture conditions also can include all timeintervals in between these exemplary periods. It is further understoodthat the time of culturing the microbial organism of the invention isfor a sufficient period of time to produce a sufficient amount ofproduct for a desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of succinate can be utilized in, forexample, fed-batch fermentation and batch separation; fed-batchfermentation and continuous separation, or continuous fermentation andcontinuous separation. Examples of batch and continuous fermentationprocedures are well known in the art.

In addition to the above fermentation procedures using the succinateproducers for continuous production of substantial quantities ofsuccinate, the succinate producers also can be, for example,simultaneously subjected to chemical synthesis procedures to convert theproduct to other compounds or the product can be separated from thefermentation culture and sequentially subjected to chemical conversionto convert the product to other compounds, if desired.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. Publ. Nos. 2002/0012939, 2003/0224363, 2004/0029149,2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466, and U.S. Pat.No. 7,127,379). Modeling analysis allows reliable predictions of theeffects on cell growth of shifting the metabolism towards more efficientproduction of succinate.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion or disruption strategies that result ingenetically stable microorganisms which overproduce the target product.Specifically, the framework examines the complete metabolic and/orbiochemical network of a microorganism in order to suggest geneticmanipulations that force the desired biochemical to become an obligatorybyproduct of cell growth. By coupling biochemical production with cellgrowth through strategically placed gene deletions or other functionalgene disruption, the growth selection pressures imposed on theengineered strains after long periods of time in a bioreactor lead toimprovements in performance as a result of the compulsory growth-coupledbiochemical production. Lastly, when gene deletions are constructedthere is a negligible possibility of the designed strains reverting totheir wild-type states because the genes selected by OptKnock are to becompletely removed from the genome. Therefore, this computationalmethodology can be used to either identify alternative pathways thatlead to biosynthesis of a desired product or used in connection with thenon-naturally occurring microbial organisms for further optimization ofbiosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat allow an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. Publ. No. 2002/0168654, International Patent Application No.PCT/US02/00660, and U.S. Publ. No. 2009/0047719.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. Publ. No.2003/0233218, and International Patent Application No. PCT/US03/18838.SimPheny® is a computational system that can be used to produce anetwork model in silico and to simulate the flux of mass, energy orcharge through the chemical reactions of a biological system to define asolution space that contains any and all possible functionalities of thechemical reactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. Publ. Nos. 2002/0012939,2003/0224363, 2004/0029149, 2004/0072723, 2003/0059792, 2002/0168654 and2004/0009466, and in U.S. Pat. No. 7,127,379. As disclosed herein, theOptKnock mathematical framework can be applied to pinpoint genedeletions leading to the growth-coupled production of a desired product.Further, the solution of the bilevel OptKnock problem provides only oneset of deletions. To enumerate all meaningful solutions, that is, allsets of knockouts leading to growth-coupled production formation, anoptimization technique, termed integer cuts, can be implemented. Thisentails iteratively solving the OptKnock problem with the incorporationof an additional constraint referred to as an integer cut at eachiteration, as discussed above.

As disclosed herein, a nucleic acid encoding a desired activity of asuccinate pathway, formaldehyde assimilation pathway, and/or methanolmetabolic pathway can be introduced into a host organism. In some cases,it can be desirable to modify an activity of a succinate pathway ormethanol metabolic pathway enzyme or protein to increase production ofsuccinate; formaldehyde, and/or reducing equivalents. For example, knownmutations that increase the activity of a protein or enzyme can beintroduced into an encoding nucleic acid molecule. Additionally,optimization methods can be applied to increase the activity of anenzyme or protein and/or decrease an inhibitory activity, for example,decrease the activity of a negative regulator.

One such optimization method is directed evolution. Directed evolutionis a powerful approach that involves the introduction of mutationstargeted to a specific gene in order to improve and/or alter theproperties of an enzyme. Improved and/or altered enzymes can beidentified through the development and implementation of sensitivehigh-throughput screening assays that allow the automated screening ofmany enzyme variants (for example, >10⁴). Iterative rounds ofmutagenesis and screening typically are performed to afford an enzymewith optimized properties. Computational algorithms that can help toidentify areas of the gene for mutagenesis also have been developed andcan significantly reduce the number of enzyme variants that need to begenerated and screened. Numerous directed evolution technologies havebeen developed (for reviews, see Hibbert et al., Biomol. Eng. 22:11-19(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical andbiotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press;Otten and Quax. Biomol. Eng. 22:1-9 (2005); and Sen et al., ApplBiochem. Biotechnol 143:212-223 (2007)) to be effective at creatingdiverse variant libraries, and these methods have been successfullyapplied to the improvement of a wide range of properties across manyenzyme classes. Enzyme characteristics that have been improved and/oraltered by directed evolution technologies include, for example:selectivity/specificity, for conversion of non-natural substrates;temperature stability, for robust high temperature processing; pHstability, for bioprocessing under lower or higher pH conditions;substrate or product tolerance, so that high product titers can beachieved; binding (K_(m)), including broadening substrate binding toinclude non-natural substrates; inhibition (K_(i)), to remove inhibitionby products, substrates, or key intermediates; activity (kcat), toincreases enzymatic reaction rates to achieve desired flux; expressionlevels, to increase protein yields and overall pathway flux; oxygenstability, for operation of air sensitive enzymes under aerobicconditions; and anaerobic activity, for operation of an aerobic enzymein the absence of oxygen.

A number of exemplary methods have been developed for the mutagenesisand diversification of genes to target desired properties of specificenzymes. Such methods are well known to those skilled in the art. Any ofthese can be used to alter and/or optimize the activity of a succinatepathway and/or a methanol metabolic pathway enzyme or protein. Suchmethods include, but are not limited to EpPCR, which introduces randompoint mutations by reducing the fidelity of DNA polymerase in PCRreactions (Pritchard et al., J. Theor. Biol. 234:497-509 (2005));Error-prone Rolling Circle Amplification (epRCA), which is similar toepPCR except a whole circular plasmid is used as the template and random6-mers with exonuclease resistant thiophosphate linkages on the last 2nucleotides are used to amplify the plasmid followed by transformationinto cells in which the plasmid is re-circularized at tandem repeats(Fujii et al., Nucleic Acids Res. 32:e145 (2004); and Fujii et al., Nat.Protocols 1:2493-2497 (2006)); DNA or Family Shuffling, which typicallyinvolves digestion of two or more variant genes with nucleases such asDnase I or EndoV to generate a pool of random fragments that arereassembled by cycles of annealing and extension in the presence of DNApolymerase to create a library of chimeric genes (Stemmer, Proc. Natl.Acad. Sci. U.S.A. 91:10747-10751 (1994); and Stemmer, Nature 370:389-391(1994)); Staggered Extension (StEP), which entails template primingfollowed by repeated cycles of 2 step PCR with denaturation and veryshort duration of annealing/extension (as short as 5 sec) (Zhao et al.,Nat. Biotechnol. 16:258-261 (1998)); Random Priming Recombination (RPR),in which random sequence primers are used to generate many short DNAfragments complementary to different segments of the template (Shao etal., Nucleic Acids Res. 26:681-683 (1998)).

Additional methods include Heteroduplex Recombination, in whichlinearized plasmid DNA is used to form heteroduplexes that are repairedby mismatch repair (Volkov et al, Nucleic Acids Res. 27:e18 (1999); andVolkov et al., Methods Enzymol. 328:456-463 (2000)); RandomChimeragenesis on Transient Templates (RACHITT), which employs Dnase Ifragmentation and size fractionation of single stranded DNA (ssDNA)(Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extensionon Truncated templates (RETT), which entails template switching ofunidirectionally growing strands from primers in the presence ofunidirectional ssDNA fragments used as a pool of templates (Lee et al.,J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide GeneShuffling (DOGS), in which degenerate primers are used to controlrecombination between molecules; (Bergquist and Gibbs, Methods Mol.Biol. 352:191-204 (2007); Bergquist et al., Biomol. Eng. 22:63-72(2005); Gibbs et al., Gene 271:13-20 (2001)); Incremental Truncation forthe Creation of Hybrid Enzymes (ITCHY), which creates a combinatoriallibrary with 1 base pair deletions of a gene or gene fragment ofinterest (Ostermeier et al., Proc. Natl. Acad. Sci. U.S.A. 96:3562-3567(1999); and Ostermeier et al., Nat. Biotechnol. 17:1205-1209 (1999));Thio-Incremental Truncation for the Creation of Hybrid Enzymes(THIO-ITCHY), which is similar to ITCHY except that phosphothioate dNTPsare used to generate truncations (Lutz et al., Nucleic Acids Res. 29:E16(2001)); SCRATCHY, which combines two methods for recombining genes,ITCHY and DNA shuffling (Lutz et al., Proc. Natl. Acad. Sci. U.S.A.98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in whichmutations made via epPCR are followed by screening/selection for thoseretaining usable activity (Bergquist et al., Biomol. Eng. 22:61-72(2005)); Sequence Saturation Mutagenesis (SeSaM), a random mutagenesismethod that generates a pool of random length fragments using randomincorporation of a phosphothioate nucleotide and cleavage, which is usedas a template to extend in the presence of “universal” bases such asinosine, and replication of an inosine-containing complement givesrandom base incorporation and, consequently, mutagenesis (Wong et al.,Biotechnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26(2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)); SyntheticShuffling, which uses overlapping oligonucleotides designed to encode“all genetic diversity in targets” and allows a very high diversity forthe shuffled progeny (Ness et al., Nat. Biotechnol. 20:1251-1255(2002)); Nucleotide Exchange and Excision Technology NexT, whichexploits a combination of dUTP incorporation followed by treatment withuracil DNA glycosylase and then piperidine to perform endpoint DNAfragmentation (Muller et al., Nucleic Acids Res. 33:e117 (2005)).

Further methods include Sequence Homology-Independent ProteinRecombination (SHIPREC), in which a linker is used to facilitate fusionbetween two distantly related or unrelated genes, and a range ofchimeras is generated between the two genes, resulting in libraries ofsingle-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460(2001)); Gene Site Saturation Mutagenesis™ (GSSM™), in which thestarting materials include a supercoiled double stranded DNA (dsDNA)plasmid containing an insert and two primers which are degenerate at thedesired site of mutations (Kretz et al., Methods Enzymol. 388:3-11(2004)); Combinatorial Cassette Mutagenesis (CCM), which involves theuse of short oligonucleotide cassettes to replace limited regions with alarge number of possible amino acid sequence alterations (Reidhaar-Olsonet al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al.Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis(CMCM), which is essentially similar to CCM and uses epPCR at highmutation rate to identify hot spots and hot regions and then extensionby CMCM to cover a defined region of protein sequence space (Reetz etal., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the MutatorStrains technique, in which conditional is mutator plasmids, utilizingthe mutD5 gene, which encodes a mutant subunit of DNA polymerase III, toallow increases of 20 to 4000-× in random and natural mutation frequencyduring selection and block accumulation of deleterious mutations whenselection is not required (Selifonova et al., Appl. Environ. Microbiol.67:3645-3649 (2001)); Low et al., J. Mol. Biol. 260:359-3680 (1996)).

Additional exemplary methods include Look-Through Mutagenesis (LTM),which is a multidimensional mutagenesis method that assesses andoptimizes combinatorial mutations of selected amino acids (Rajpal etal., Proc. Natl. Acad. Sci. U.S.A. 102:8466-8471 (2005)); GeneReassembly, which is a DNA shuffling method that can be applied tomultiple genes at one time or to create a large library of chimeras(multiple mutations) of a single gene (Tunable GeneReassembly™ (TGR™)Technology supplied by Verenium Corporation), in Silico Protein DesignAutomation (PDA), which is an optimization algorithm that anchors thestructurally defined protein backbone possessing a particular fold, andsearches sequence space for amino acid substitutions that can stabilizethe fold and overall protein energetics, and generally works mosteffectively on proteins with known three-dimensional structures (Hayeset al., Proc. Natl. Acad. Sci. U.S.A. 99:15926-15931 (2002)); andIterative Saturation Mutagenesis (ISM), which involves using knowledgeof structure/function to choose a likely site for enzyme improvement,performing saturation mutagenesis at chosen site using a mutagenesismethod such as Stratagene QuikChange (Stratagene; San Diego Calif.),screening/selecting for desired properties, and, using improvedclone(s), starting over at another site and continue repeating until adesired activity is achieved (Reetz et al., Nat. Protocols 2:891-903(2007); and Reetz et al., Angew. Chem. Int. Ed Engl. 45:7745-7751(2006)).

Any of the aforementioned methods for mutagenesis can be used alone orin any combination. Additionally, any one or combination of the directedevolution methods can be used in conjunction with adaptive evolutiontechniques, as described herein.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties, includingGenBank and GI number publications, are hereby incorporated by referencein this application in order to more fully describe the state of the artto which this invention pertains. Although the invention has beendescribed with reference to the examples provided above, it should beunderstood that various modifications can be made without departing fromthe spirit of the invention.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

4. EXAMPLES 4.1 Example I Production of Reducing Equivalents Via aMethanol Metabolic Pathway

Exemplary methanol metabolic pathways are provided in FIG. 1.

FIG. 1, Step A—Methanol Methyltransferase

A complex of 3-methyltransferase proteins, denoted MtaA, MtaB, and MtaC,perform the desired methanol methyltransferase activity (Sauer et al.,Eur. J. Biochem. 243:670-677 (1997); Naidu and Ragsdale, J. Bacteriol.183:3276-3281 (2001); Tallant and Krzycki, J. Biol. Chem. 276:4485-4493(2001); Tallant and Krzycki, J. Bacteriol. 179:6902-6911 (1997); Tallantand Krzycki, J. Bacteriol. 178:1295-1301 (1996); Ragsdale, S. W., Crit.Rev. Biochem. Mol. Biol. 39:165-195 (2004)).

MtaB is a zinc protein that can catalyze the transfer of a methyl groupfrom methanol to MtaC, a corrinoid protein. Exemplary genes encodingMtaB and MtaC can be found in methanogenic archaea such asMethanosarcina barkeri (Maeder et al., J. Bacteriol. 188:7922-7931(2006) and Methanosarcina acetivorans (Galagan et al., Genome Res.12:532-542 (2002), as well as the acetogen, Moorella thermoacetica (Daset al., Proteins 67:167-176 (2007). In general, the MtaB and MtaC genesare adjacent to one another on the chromosome as their activities aretightly interdependent. The protein sequences of various MtaB and MtaCencoding genes in M. barkeri, M. acetivorans, and M. thermoaceticum canbe identified by their following GenBank accession numbers.

Protein GenBank ID GI number Organism MtaB1 YP_304299 73668284Methanosarcina barkeri MtaC1 YP_304298 73668283 Methanosarcina barkeriMtaB2 YP_307082 73671067 Methanosarcina barkeri MtaC2 YP_307081 73671066Methanosarcina barkeri MtaB3 YP_304612 73668597 Methanosarcina barkeriMtaC3 YP_304611 73668596 Methanosarcina barkeri MtaB1 NP_615421 20089346Methanosarcina acetivorans MtaB1 NP_615422 20089347 Methanosarcinaacetivorans MtaB2 NP_619254 20093179 Methanosarcina acetivorans MtaC2NP_619253 20093178 Methanosarcina acetivorans MtaB3 NP_616549 20090474Methanosarcina acetivorans MtaC3 NP_616550 20090475 Methanosarcinaacetivorans MtaB YP_430066 83590057 Moorella thermoacetica MtaCYP_430065 83590056 Moorella thermoacetica MtaA YP_430064 83590056Moorella thermoacetica

MtaB is a zinc protein that can catalyze the transfer of a methyl groupfrom methanol to MtaC, a corrinoid protein. Exemplary genes encodingMtaB and MtaC can be found in methanogenic archaea such asMethanosarcina barkeri (Maeder et al., J. Bacteriol. 188:7922-7931(2006) and Methanosarcina acetivorans (Galagan et al., Genome Res.12:532-542 (2002), as well as the acetogen, Moorella thermoacetica (Daset al., Proteins 67:167-176 (2007). In general, the MtaB and MtaC genesare adjacent to one another on the chromosome as their activities aretightly interdependent. The protein sequences of various MtaB and MtaCencoding genes in M. barkeri, M. acetivorans, and M. thermoaceticum canbe identified by their following GenBank accession numbers.

The MtaB1 and MtaC1 genes, YP_304299 and YP_304298, from M. barkeri werecloned into E. coli and sequenced (Sauer et al., Eur. J. Biochem.243:670-677 (1997)). The crystal structure of this methanol-cobalaminmethyltransferase complex is also available (Hagemeier et al., Proc.Natl. Acad. Sci. U.S.A. 103:18917-18922 (2006)). The MtaB genes,YP_307082 and YP_304612, in M. barkeri were identified by sequencehomology to YP_304299. In general, homology searches are an effectivemeans of identifying methanol methyltransferases because MtaB encodinggenes show little or no similarity to methyltransferases that act onalternative substrates such as trimethylamine, dimethylamine,monomethylamine, or dimethylsulfide. The MtaC genes, YP_307081 andYP_304611 were identified based on their proximity to the MtaB genes andalso their homology to YP_304298. The three sets of MtaB and MtaC genesfrom M. acetivorans have been genetically, physiologically, andbiochemically characterized (Pritchett and Metcalf, Mol. Microbiol.56:1183-1194 (2005)). Mutant strains lacking two of the sets were ableto grow on methanol, whereas a strain lacking all three sets of MtaB andMtaC genes sets could not grow on methanol. This suggests that each setof genes plays a role in methanol utilization. The M. thermoacetica MtaBgene was identified based on homology to the methanogenic MtaB genes andalso by its adjacent chromosomal proximity to the methanol-inducedcorrinoid protein, MtaC, which has been crystallized (Zhou et al., ActaCrystallogr. Sect. F. Struct. Biol. Cyrst. Commun. 61:537-540 (2005) andfurther characterized by Northern hybridization and Western Blotting((Das et al., Proteins 67:167-176 (2007)).

MtaA is zinc protein that catalyzes the transfer of the methyl groupfrom MtaC to either Coenzyme M in methanogens or methyltetrahydrofolatein acetogens. MtaA can also utilize methylcobalamin as the methyl donor.Exemplary genes encoding MtaA can be found in methanogenic archaea suchas Methanosarcina barkeri (Maeder et al., J. Bacteriol. 188:7922-7931(2006) and Methanosarcina acetivorans (Galagan et al., Genome Res.12:532-542 (2002), as well as the acetogen, Moorella thermoacetica ((Daset al., Proteins 67:167-176 (2007)). In general, MtaA proteins thatcatalyze the transfer of the methyl group from CH₃-MtaC are difficult toidentify bioinformatically as they share similarity to other corrinoidprotein methyltransferases and are not oriented adjacent to the MtaB andMtaC genes on the chromosomes. Nevertheless, a number of MtaA encodinggenes have been characterized. The protein sequences of these genes inM. barkeri and M. acetivorans can be identified by the following GenBankaccession numbers.

Protein GenBank ID GI number Organism MtaA YP_304602 73668587Methanosarcina barkeri MtaA1 NP_619241 20093166 Methanosarcinaacetivorans MtaA2 NP_616548 20090473 Methanosarcina acetivorans

MtaA is zinc protein that catalyzes the transfer of the methyl groupfrom MtaC to either Coenzyme M in methanogens or methyltetrahydrofolatein acetogens. MtaA can also utilize methylcobalamin as the methyl donor.Exemplary genes encoding MtaA can be found in methanogenic archaea suchas Methanosarcina barkeri (Maeder et al., J. Bacteriol. 188:7922-7931(2006) and Methanosarcina acetivorans (Galagan et al., Genome Res.12:532-542 (2002), as well as the acetogen, Moorella thermoacetica ((Daset al., Proteins 67:167-176 (2007)). In general, MtaA proteins thatcatalyze the transfer of the methyl group from CH₃-MtaC are difficult toidentify bioinformatically as they share similarity to other corrinoidprotein methyltransferases and are not oriented adjacent to the MtaB andMtaC genes on the chromosomes. Nevertheless, a number of MtaA encodinggenes have been characterized. The protein sequences of these genes inM. barkeri and M. acetivorans can be identified by the following GenBankaccession numbers.

The MtaA gene, YP_304602, from M. barkeri was cloned, sequenced, andfunctionally overexpressed in E. coli (Harms and Thauer, Eur. J.Biochem. 235:653-659 (1996)). In M. acetivorans, MtaA1 is required forgrowth on methanol, whereas MtaA2 is dispensable even though methaneproduction from methanol is reduced in MtaA2 mutants (Bose et al., J.Bacteriol. 190:4017-4026 (2008)). There are multiple additional MtaAhomologs in M. barkeri and M. acetivorans that are as yetuncharacterized, but may also catalyze corrinoid proteinmethyltransferase activity.

Putative MtaA encoding genes in M. thermoacetica were identified bytheir sequence similarity to the characterized methanogenic MtaA genes.Specifically, three M. thermoacetica genes show high homology (>30%sequence identity) to YP_304602 from M. barkeri. Unlike methanogenicMtaA proteins that naturally catalyze the transfer of the methyl groupfrom CH₃-MtaC to Coenzyme M, an M. thermoacetica MtaA is likely totransfer the methyl group to methyltetrahydrofolate given the similarroles of methyltetrahydrofolate and Coenzyme M in methanogens andacetogens, respectively. The protein sequences of putative MtaA encodinggenes from M. thermoacetica can be identified by the following GenBankaccession numbers.

Protein GenBank ID GI number Organism MtaA YP_430937 83590928 Moorellathermoacetica MtaA YP_431175 83591166 Moorella thermoacetica MtaAYP_430935 83590926 Moorella thermoacetica MtaA YP_430064 83590056Moorella thermoacetica

FIG. 1, Step B—Methylenetetrahydrofolate Reductase

The conversion of methyl-THF to methylenetetrahydrofolate is catalyzedby methylenetetrahydrofolate reductase. In M. thermoacetica, this enzymeis oxygen-sensitive and contains an iron-sulfur cluster (Clark andLjungdahl, J. Biol. Chem. 259:10845-10849 (1984). This enzyme is encodedby metF in E. coli (Sheppard et al., J. Bacteriol. 181:718-725 (1999)and CHY_1233 in C. hydrogenoformans (Wu et al., PLoS Genet. 1:e65(2005). The M. thermoacetica genes, and its C. hydrogenoformanscounterpart, are located near the CODH/ACS gene cluster, separated byputative hydrogenase and heterodisulfide reductase genes. Someadditional gene candidates found bioinformatically are listed below. InAcetobacterium woodii metF is coupled to the Rnf complex through RnfC2(Poehlein et al, PLoS One. 7:e33439). Homologs of RnfC are found inother organisms by blast search. The Rnf complex is known to be areversible complex (Fuchs (2011) Annu. Rev. Microbiol. 65:631-658).

Protein GenBank ID GI number Organism Moth_1191 YP_430048.1 83590039Moorella thermoacetica Moth_1192 YP_430049.1 83590040 Moorellathermoacetica metF NP_418376.1 16131779 Escherichia coli CHY_1233YP_360071.1 78044792 Carboxydothermus hydrogenoformans CLJU_c37610YP_003781889.1 300856905 Clostridium ljungdahlii DSM13528DesfrDRAFT_3717 ZP_07335241.1 303248996 Desulfovibrio fructosovorans JJCcarbDRAFT_2950 ZP_05392950.1 255526026 Clostridium carboxidivorans P7Cce174_010100023124 ZP_07633513.1 307691067 Clostridium cellulovorans743B Cphy_3110 YP_001560205.1 160881237 Clostridium phytofermentans ISDg

FIG. 1, Steps C and D—Methylenetetrahydrofolate Dehydrogenase,Methenyltetrahydrofolate Cyclohydrolase

In M. thermoacetica, E. coli, and C. hydrogenoformans,methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolatedehydrogenase are carried out by the bi-functional gene products ofMoth_1516, folD, and CHY_1878, respectively (Pierce et al., Environ.Microbiol. 10:2550-2573 (2008); Wu et al., PLoS Genet. 1:e65 (2005);D'Ari and Rabinowitz, J. Biol. Chem. 266:23953-23958 (1991)). A homologexists in C. carboxidivorans P7. Several other organisms also encode forthis bifunctional protein as tabulated below.

Protein GenBank ID GI number Organism Moth_l516 YP_430368.1 83590359Moorella thermoacetica folD NP_415062.1 16128513 Escherichia coliCHY_1878 YP_360698.1 78044829 Carboxydothermus hydrogenoformansCcarbDRAFT_2948 ZP_05392948.1 255526024 Clostridium carboxidivorans P7folD ADK16789.1 300437022 Clostridium ljungdahlii DSM13528 folD-2NP_951919.1 39995968 Geobacter sulfurreducens PCA folD YP_725874.1113867385 Ralstonia eutropha H16 folD NP_348702.1 15895353 Clostridiumacetobutylicum ATCC 824 folD YP_696506.1 110800457 Clostridiumperfringens MGA3_09460 EIJ83438.1 387591119 Bacillus methanolicus MGA3PB1_14689 ZP_10132349.1 387929672 Bacillus methanolicus PB1

FIG. 1, Step E—Formyltetrahydrofolate Deformylase

This enzyme catalyzes the hydrolysis of 10-formyltetrahydrofolate(formyl-THF) to THF and formate. In E. coli, this enzyme is encoded bypurU and has been overproduced, purified, and characterized (Nagy, etal., J. Bacteria 3:1292-1298 (1995)). Homologs exist in Corynebacteriumsp. U-96 (Suzuki, et al., Biosci. Biotechnol. Biochem. 69(5):952-956(2005)), Corynebacterium glutamicum ATCC 14067, Salmonella enterica, andseveral additional organisms.

Protein GenBank ID GI number Organism purU AAC74314.1 1787483Escherichia coli K-12 MG1655 purU BAD97821.1 63002616 Corynebacteriumsp. U-96 purU EHE84645.1 354511740 Corynebacterium glutamicum ATCC 14067purU NP_460715.1 16765100 Salmonella enterica subsp. enterica serovarTyphimurium str. LT2

FIG. 1, Step F—Formyltetrahydrofolate Synthetase

Formyltetrahydrofolate synthetase ligates formate to tetrahydrofolate atthe expense of one ATP. This reaction is catalyzed by the gene productof Moth_0109 in M. thermoacetica (O'brien et al., Experientia Suppl.26:249-262 (1976); Lovell et al., Arch. Microbiol. 149:280-285 (1988);Lovell et al., Biochemistry 29:5687-5694 (1990)), FHS in Clostridiumacidurici (Whitehead and Rabinowitz, J. Bacteriol. 167:203-209 (1986);Whitehead and Rabinowitz, J. Bacteriol. 170:3255-3261 (1988), andCHY_2385 in C. hydrogenoformans (Wu et al., PLoS Genet. 1:e65 (2005).Homologs exist in C. carboxidivorans P7. This enzyme is found in severalother organisms as listed below.

Protein GenBank ID GI number Organism Moth_0109 YP_428991.1 83588982Moorella thermoacetica CHY_2385 YP_361182.1 78045024 Carboxydothermushydrogenoformans FHS P13419.1 120562 Clostridium aciduriciCcarbDRAFT_1913 ZP_05391913.1 255524966 Clostridium carboxidivorans P7CcarbDRAFT_2946 ZP_05392946.1 255526022 Clostridium carboxidivorans P7Dhaf_0555 ACL18622.1 219536883 Desulfitobacterium hafniense FhsYP_001393842.1 153953077 Clostridium kluyveri DSM 555 Fhs YP_003781893.1300856909 Clostridium ljungdahlii DSM 13528 MGA3_08300 EIJ83208.1387590889 Bacillus methanolicus MGA3 PB1_13509 ZP_10132113.1 387929436Bacillus methanolicus PB1

FIG. 1, Step G—Formate Hydrogen Lyase

A formate hydrogen lyase enzyme can be employed to convert formate tocarbon dioxide and hydrogen. An exemplary formate hydrogen lyase enzymecan be found in Escherichia coli. The E. coli formate hydrogen lyaseconsists of hydrogenase 3 and formate dehydrogenase-H (Maeda et al.,Appl Microbiol Biotechnol 77:879-890 (2007)). It is activated by thegene product of fhlA. (Maeda et al., Appl Microbiol Biotechnol77:879-890 (2007)). The addition of the trace elements, selenium, nickeland molybdenum, to a fermentation broth has been shown to enhanceformate hydrogen lyase activity (Soini et al., Microb. Cell Fact. 7:26(2008)). Various hydrogenase 3, formate dehydrogenase andtranscriptional activator genes are shown below.

Protein GenBank ID GI number Organism hycA NP_417205 16130632Escherichia coli K-12 MG1655 hycB NP_417204 16130631 Escherichia coliK-12 MG1655 hycC NP_417203 16130630 Escherichia coli K-12 MG1655 hycDNP_417202 16130629 Escherichia coli K-12 MG1655 hycE NP_417201 16130628Escherichia coli K-12 MG1655 hycF NP_417200 16130627 Escherichia coliK-12 MG1655 hycG NP_417199 16130626 Escherichia coli K-12 MG1655 hycHNP_417198 16130625 Escherichia coli K-12 MG1655 hycI NP_417197 16130624Escherichia coli K-12 MG1655 fdhF NP_418503 16131905 Escherichia coliK-12 MG1655 fhlA NP_417211 16130638 Escherichia coli K-12 MG1655

A formate hydrogen lyase enzyme also exists in the hyperthermophilicarchaeon, Thermococcus litoralis (Takacs et al., BMC. Microbiol 8:88(2008)).

Protein GenBank ID GI number Organism mhyC ABW05543 157954626Thermococcus litoralis mhyD ABW05544 157954627 Thermococcus litoralismhyE ABW05545 157954628 Thermococcus litoralis myhF ABW05546 157954629Thermococcus litoralis myhG ABW05547 157954630 Thermococcus litoralismyhH ABW05548 157954631 Thermococcus litoralis fdhA AAB94932 2746736Thermococcus litoralis fdhB AAB94931 157954625 Thermococcus litoralis

Additional formate hydrogen lyase systems have been found in Salmonellatyphimurium, Klebsiella pneumoniae, Rhodospirillum rubrum,Methanobacterium formicicum (Vardar-Schara et al., MicrobialBiotechnology 1:107-125 (2008)).

FIG. 1, Step H—Hydrogenase

Hydrogenase enzymes can convert hydrogen gas to protons and transferelectrons to acceptors such as ferredoxins, NAD+, or NADP+. Ralstoniaeutropha H16 uses hydrogen as an energy source with oxygen as a terminalelectron acceptor. Its membrane-bound uptake [NiFe]-hydrogenase is an“O2-tolerant” hydrogenase (Cracknell, et al. Proc Nat Acad Sci, 106(49)20681-20686 (2009)) that is periplasmically-oriented and connected tothe respiratory chain via a b-type cytochrome (Schink and Schlegel,Biochim. Biophys. Acta, 567, 315-324 (1979); Bernhard et al., Eur. J.Biochem. 248, 179-186 (1997)). R. eutropha also contains an O₂-tolerantsoluble hydrogenase encoded by the Hox operon which is cytoplasmic anddirectly reduces NAD+ at the expense of hydrogen (Schneider andSchlegel, Biochim. Biophys. Acta 452, 66-80 (1976); Burgdorf, J Bact.187(9) 3122-3132 (2005)). Soluble hydrogenase enzymes are additionallypresent in several other organisms including Geobacter sulfurreducens(Coppi, Microbiology 151, 1239-1254 (2005)), Synechocystis str. PCC 6803(Germer, J. Biol. Chem., 284(52), 36462-36472 (2009)), and Thiocapsaroseopersicina (Rakhely, Appl. Environ. Microbiol. 70(2) 722-728(2004)). The Synechocystis enzyme is capable of generating NADPH fromhydrogen. Overexpression of both the Hox operon from Synechocystis str.PCC 6803 and the accessory genes encoded by the Hyp operon from Nostocsp. PCC 7120 led to increased hydrogenase activity compared toexpression of the Hox genes alone (Germer, J. Biol. Chem. 284(52),36462-36472 (2009)).

Protein GenBank ID GI Number Organism HoxF NP_942727.1 38637753Ralstonia eutropha H16 HoxU NP_942728.1 38637754 Ralstonia eutropha H16HoxY NP_942729.1 38637755 Ralstonia eutropha H16 HoxH NP_942730.138637756 Ralstonia eutropha H16 HoxW NP_942731.1 38637757 Ralstoniaeutropha H16 HoxI NP_942732.1 38637758 Ralstonia eutropha H16 HoxENP_953767.1 39997816 Geobacter sulfurreducens HoxF NP_953766.1 39997815Geobacter sulfurreducens HoxU NP_953765.1 39997814 Geobactersulfurreducens HoxY NP_953764.1 39997813 Geobacter sulfurreducens HoxHNP_953763.1 39997812 Geobacter sulfurreducens GSU27I7 NP_953762.139997811 Geobacter sulfurreducens HoxE NP_441418.1 16330690Synechocystis str. PCC 6803 HoxF NP_441417.1 16330689 Synechocystis str.PCC 6803 Unknown NP_441416.1 16330688 Synechocystis str. PCC 6803function HoxU NP_441415.1 16330687 Synechocystis str. PCC 6803 HoxYNP_441414.1 16330686 Synechocystis str. PCC 6803 Unknown NP_441413.116330685 Synechocystis str. PCC 6803 function Unknown NP_441412.116330684 Synechocystis str. PCC 6803 function HoxH NP_441411.1 16330683Synechocystis str. PCC 6803 HypF NP_484737.1 17228189 Nostoc sp. PCC7120 HypC NP_484738.1 17228190 Nostoc sp. PCC 7120 HypD NP_484739.117228191 Nostoc sp. PCC 7120 Unknown NP_484740.1 17228192 Nostoc sp. PCC7120 function HypE NP_484741.1 17228193 Nostoc sp. PCC 7120 HypANP_484742.1 17228194 Nostoc sp. PCC 7120 HypB NP_484743.1 17228195Nostoc sp. PCC 7120 Hox1E AAP50519.1 37787351 Thiocapsa roseopersicinaHox1F AAP50520.1 37787352 Thiocapsa roseopersicina Hox1U AAP50521.137787353 Thiocapsa roseopersicina Hox1Y AAP50522.1 37787354 Thiocapsaroseopersicina Hox1H AAP50523.1 37787355 Thiocapsa roseopersicina

The genomes of E. coli and other enteric bacteria encode up to fourhydrogenase enzymes (Sawers, G., Antonie Van Leeuwenhoek 66:57-88(1994); Sawers et al., J Bacteria 164:1324-1331 (1985); Sawers andBoxer, Eur. J Biochem. 156:265-275 (1986); Sawers et al., J. Bacteriol.168:398-404 (1986)). Given the multiplicity of enzyme activities E. colior another host organism can provide sufficient hydrogenase activity tosplit incoming molecular hydrogen and reduce the corresponding acceptor.Endogenous hydrogen-lyase enzymes of E. coli include hydrogenase 3, amembrane-bound enzyme complex using ferredoxin as an acceptor, andhydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4are encoded by the hyc and hyf gene clusters, respectively. Hydrogenaseactivity in E. coli is also dependent upon the expression of the hypgenes whose corresponding proteins are involved in the assembly of thehydrogenase complexes (Jacobi et al., Arch. Microbiol 158:444-451(1992); Rangarajan et al., J Bacteria 190:1447-1458 (2008)). The M.thermoacetica and Clostridium ljungdahii hydrogenases are suitable for ahost that lacks sufficient endogenous hydrogenase activity. M.thermoacetica and C. ljungdahli can grow with CO₂ as the exclusivecarbon source indicating that reducing equivalents are extracted from H₂to enable acetyl-CoA synthesis via the Wood-Ljungdahl pathway (Drake, H.L., J Bacteriol. 150:702-709 (1982); Drake and Daniel, Res Microbiol155:869-883 (2004); Kellum and Drake, J Bacteriol. 160:466-469 (1984)).M. thermoacetica has homologs to several hyp, hyc, and hyf genes from E.coli. These protein sequences encoded for by these genes are identifiedby the following GenBank accession numbers. In addition, several geneclusters encoding hydrogenase functionality are present in M.thermoacetica and C. ljungdahli (see for example US 2012/0003652).

Protein GenBank ID GI Number Organism HypA NP_417206 16130633Escherichia coli HypB NP_417207 16130634 Escherichia coli HypC NP_41720816130635 Escherichia coil HypD NP_417209 16130636 Escherichia coli HypENP_417210 226524740 Escherichia coli HypF NP_417192 16130619 Escherichiacoil HycA NP_417205 16130632 Escherichia coli HycB NP_417204 16130631Escherichia coil HycC NP_417203 16130630 Escherichia coli HycD NP_41720216130629 Escherichia coli HycE NP_417201 16130628 Escherichia coli HycFNP_417200 16130627 Escherichia coil HycG NP_417199 16130626 Escherichiacoil HycH NP_417198 16130625 Escherichia coli HycI NP_417197 16130624Escherichia coli HyfA NP_416976 90111444 Escherichia coil HyfB NP_41697716130407 Escherichia coil HyfC NP_416978 90111445 Escherichia coli HyfDNP_416979 16130409 Escherichia coli HyfE NP_416980 16130410 Escherichiacoli HyfF NP_416981 16130411 Escherichia coil HyfG NP_416982 16130412Escherichia coli HyfH NP_416983 16130413 Escherichia coli HyfI NP_41698416130414 Escherichia coli HyfJ NP_416985 90111446 Escherichia coli HyfRNP_416986 90111447 Escherichia coli

Proteins in M. thermoacetica whose genes are homologous to the E. colihydrogenase genes are shown below.

Protein GenBank ID GI Number Organism Moth_2175 YP_431007 83590998Moorella thermoacetica Moth_2176 YP_431008 83590999 Moorellathermoacetica Moth_2177 YP_431009 83591000 Moorella thermoaceticaMoth_2178 YP_431010 83591001 Moorella thermoacetica Moth_2179 YP_43101183591002 Moorella thermoacetica Moth_2180 YP_431012 83591003 Moorellathermoacetica Moth_2181 YP_431013 83591004 Moorella thermoaceticaMoth_2182 YP_431014 83591005 Moorella thermoacetica Moth_2183 YP_43101583591006 Moorella thermoacetica Moth_2184 YP_431016 83591007 Moorellathermoacetica Moth_2185 YP_431017 83591008 Moorella thermoaceticaMoth_2186 YP_431018 83591009 Moorella thermoacetica Moth_2187 YP_43101983591010 Moorella thermoacetica Moth_2188 YP_431020 83591011 Moorellathermoacetica Moth_2189 YP_431021 83591012 Moorella thermoaceticaMoth_2190 YP_431022 83591013 Moorella thermoacetica Moth_2191 YP_43102383591014 Moorella thermoacetica Moth_2192 YP_431024 83591015 Moorellathermoacetica Moth_0439 YP_429313 83589304 Moorella thermoaceticaMoth_0440 YP_429314 83589305 Moorella thermoacetica Moth_0441 YP_42931583589306 Moorella thermoacetica Moth_0442 YP_429316 83589307 Moorellathermoacetica Moth_0809 YP_429670 83589661 Moorella thermoaceticaMoth_0810 YP_429671 83589662 Moorella thermoacetica Moth_0811 YP_42967283589663 Moorella thermoacetica Moth_0812 YP_429673 83589664 Moorellathermoacetica Moth_0814 YP_429674 83589665 Moorella thermoaceticaMoth_0815 YP_429675 83589666 Moorella thermoacetica Moth_0816 YP_42967683589667 Moorella thermoacetica Moth_1193 YP_430050 83590041 Moorellathermoacetica Moth_1194 YP_430051 83590042 Moorella thermoaceticaMoth_1195 YP_430052 83590043 Moorella thermoacetica Moth_1196 YP_43005383590044 Moorella thermoacetica Moth_1717 YP_430562 83590553 Moorellathermoacetica Moth_1718 YP_430563 83590554 Moorella thermoaceticaMoth_1719 YP_430564 83590555 Moorella thermoacetica Moth_1883 YP_43072683590717 Moorella thermoacetica Moth_1884 YP_430727 83590718 Moorellathermoacetica Moth_1885 YP_430728 83590719 Moorella thermoaceticaMoth_1886 YP_430729 83590720 Moorella thermoacetica Moth_1887 YP_43073083590721 Moorella thermoacetica Moth_1888 YP_430731 83590722 Moorellathermoacetica Moth_1452 YP_430305 83590296 Moorella thermoaceticaMoth_1453 YP_430306 83590297 Moorella thermoacetica Moth_1454 YP_43030783590298 Moorella thermoacetica

Genes encoding hydrogenase enzymes from C. ljungdahli are shown below.

Protein GenBank ID GI Number Organism CLJU_c20290 ADK15091.1 300435324Clostridium ljungdahli CLJU_c07030 ADK13773.1 300434006 Clostridiumljungdahli CLJU_c07040 ADK13774.1 300434007 Clostridium ljungdahliCLJU_c07050 ADK13775.1 300434008 Clostridium ljungdahli CLJU_c07060ADK13776.1 300434009 Clostridium ljungdahli CLJU_c07070 ADK13777.1300434010 Clostridium ljungdahli CLJU_c07080 ADK13778.1 300434011Clostridium ljungdahli CLJU_c14730 ADK14541.1 300434774 Clostridiumljungdahli CLJU_c14720 ADK14540.1 300434773 Clostridium ljungdahliCLJU_c14710 ADK14539.1 300434772 Clostridium ljungdahli CLJU_c14700ADK14538.1 300434771 Clostridium ljungdahli CLJU_c28670 ADK15915.1300436148 Clostridium ljungdahli CLJU_c28660 ADK15914.1 300436147Clostridium ljungdahli CLJU_c28650 ADK15913.1 300436146 Clostridiumljungdahli CLJU_c28640 ADK15912.1 300436145 Clostridium ljungdahli

In some cases, hydrogenase encoding genes are located adjacent to aCODH. In Rhodospirillum rubrum, the encoded CODH/hydrogenase proteinsform a membrane-bound enzyme complex that has been indicated to be asite where energy, in the form of a proton gradient, is generated fromthe conversion of CO and H₂O to CO₂ and H₂ (Fox et al., J Bacteria178:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and itsadjacent genes have been proposed to catalyze a similar functional rolebased on their similarity to the R. rubrum CODH/hydrogenase gene cluster(Wu et al., PLoS Genet. 1:e65 (2005)). The C. hydrogenoformans CODH-Iwas also shown to exhibit intense CO oxidation and CO₂ reductionactivities when linked to an electrode (Parkin et al., J Am. Chem. Soc.129:10328-10329 (2007)).

Protein GenBank ID GI Number Organism CooL AAC45118 1515468Rhodospirillum rubrum CooX AAC45119 1515469 Rhodospirillum rubrum CooUAAC45120 1515470 Rhodospirillum rubrum CooH AAC45121 1498746Rhodospirillum rubrum CooF AAC45122 1498747 Rhodospirillum rubrum CODHAAC45123 1498748 Rhodospirillum rubrum (CooS) CooC AAC45124 1498749Rhodospirillum rubrum CooT AAC45125 1498750 Rhodospirillum rubrum CooJAAC45126 1498751 Rhodospirillum rubrum CODH-I YP_360644 78043418Carboxydothermus (CooS-I) hydrogenoformans CooF YP_360645 78044791Carboxydothermus hydrogenoformans HypA YP_360646 78044340Carboxydothermus hydrogenoformans CooH YP_360647 78043871Carboxydothermus hydrogenoformans CooU YP_360648 78044023Carboxydothermus hydrogenoformans CooX YP_360649 78043124Carboxydothermus hydrogenoformans CooL YP_360650 78043938Carboxydothermus hydrogenoformans CooK YP_360651 78044700Carboxydothermus hydrogenoformans CooM YP_360652 78043942Carboxydothermus hydrogenoformans CooC YP_360654.1 78043296Carboxydothermus hydrogenoformans CooA-1 YP_360655.1 78044021Carboxydothermus hydrogenoformans

Some hydrogenase and CODH enzymes transfer electrons to ferredoxins.Ferredoxins are small acidic proteins containing one or more iron-sulfurclusters that function as intracellular electron carriers with a lowreduction potential. Reduced ferredoxins donate electrons toFe-dependent enzymes such as ferredoxin-NADP⁺ oxidoreductase,pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxinoxidoreductase (OFOR). The H. thermophiles gene fdxl encodes a[4Fe-4S]-type ferredoxin that is required for the reversiblecarboxylation of 2-oxoglutarate and pyruvate by OFOR and PFOR,respectively (Yamamoto et al., Extremophiles 14:79-85 (2010)). Theferredoxin associated with the Sulfolobus solfataricus2-oxoacid:ferredoxin reductase is a monomeric dicluster [3Fe-4S][4Fe-4S]type ferredoxin (Park et al. 2006). While the gene associated with thisprotein has not been fully sequenced, the N-terminal domain shares 93%homology with the zfx ferredoxin from S. acidocaldarius. The E. coligenome encodes a soluble ferredoxin of unknown physiological function,fdx. Some evidence indicates that this protein can function iniron-sulfur cluster assembly (Takahashi and Nakamura, 1999). Additionalferredoxin proteins have been characterized in Helicobacter pylori(Mukhopadhyay et al. 2003) and Campylobacter jejuni (van Vliet et al.2001). A 2Fe-2S ferredoxin from Clostridium pasteurianum has been clonedand expressed in E. coli (Fujinaga and Meyer, Biochemical andBiophysical Research Communications, 192(3): (1993)). Acetogenicbacteria such as Moorella thermoacetica, Clostridium carboxidivorans P7,Clostridium ljungdahli and Rhodospirillum rubrum are predicted to encodeseveral ferredoxins, listed below.

GI Protein GenBank ID Number Organism fdxl BAE02673.1 68163284Hydrogenobacter thermophilus M11214.1 AAA83524.1 144806 Clostridiumpasteurianum Zfx AAY79867.1 68566938 Sulfolobus acidocalarius FdxAAC75578.1 1788874 Escherichia coli hp_0277 AAD07340.1 2313367Helicobacter pylori fdxA CAL34484.1 112359698 Campylobacter jejuniMoth_0061 ABC18400.1 83571848 Moorella thermoacetica Moth_1200ABC19514.1 83572962 Moorella thermoacetica Moth_1888 ABC20188.1 83573636Moorella thermoacetica Moth_2112 ABC20404.1 83573852 Moorellathermoacetica Moth_1037 ABC19351.1 83572799 Moorella thermoaceticaCcarbDRAFT_4383 ZP_05394383.1 255527515 Clostridium carboxidivorans P7CcarbDRAFT_2958 ZP_05392958.1 255526034 Clostridium carboxidivorans P7CcarbDRAFT_2281 ZP_05392281.1 255525342 Clostridium carboxidivorans P7CcarbDRAFT_5296 ZP_05395295.1 255528511 Clostridium carboxidivorans P7CcarbDRAFT_1615 ZP_05391615.1 255524662 Clostridium carboxidivorans P7CcarbDRAFT_1304 ZP_05391304.1 255524347 Clostridium carboxidivorans P7cooF AAG29808.1 11095245 Carboxydothermus hydrogenoformans fdxNCAA35699.1 46143 Rhodobacter capsulatus Rru_A2264 ABC23064.1 83576513Rhodospirillum rubrum Rru_A1916 ABC22716.1 83576165 Rhodospirillumrubrum Rru_A2026 ABC22826.1 83576275 Rhodospirillum rubrum cooFAAC45122.1 1498747 Rhodospirillum rubrum fdxN AAA26460.1 152605Rhodospirillum rubrum Alvin_2884 ADC63789.1 288897953 Allochromatiumvinosum DSM 180 Fdx YP_002801146.1 226946073 Azotobacter vinelandii DJCKL_3790 YP_001397146.1 153956381 Clostridium kluyveri DSM 555 fer1NP_949965.1 39937689 Rhodopseudomonas palustris CGA009 Fdx CAA12251.13724172 Thauera aromatica CHY_2405 YP_361202.1 78044690 Carboxydothermushydrogenoformans Fer YP_359966.1 78045103 Carboxydothermushydrogenoformans Fer AAC83945.1 1146198 Bacillus subtilis fdx1NP_249053.1 15595559 Pseudomonas aeruginosa PA01 yfhL AP_003148.189109368 Escherichia coli K-12 CLJU_c00930 ADK13195.1 300433428Clostridium ljungdahli CLJU_c00010 ADK13115.1 300433348 Clostridiumljungdahli CLJU_c01820 ADK13272.1 300433505 Clostridium ljungdahliCLJU_c17980 ADK14861.1 300435094 Clostridium ljungdahli CLJU_c17970ADK14860.1 300435093 Clostridium ljungdahli CLJU_c22510 ADK15311.1300435544 Clostridium ljungdahli CLJU_c26680 ADK15726.1 300435959Clostridium ljungdahli CLJU_c29400 ADK15988.1 300436221 Clostridiumljungdahli

Ferredoxin oxidoreductase enzymes transfer electrons from ferredoxins orflavodoxins to NAD(P)H. Two enzymes catalyzing the reversible transferof electrons from reduced ferredoxins to NAD(P)+ are ferredoxin:NAD+oxidoreductase (EC 1.18.1.3) and ferredoxin:NADP+ oxidoreductase (FNR,EC 1.18.1.2). Ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) has anoncovalently bound FAD cofactor that facilitates the reversibletransfer of electrons from NADPH to low-potential acceptors such asferredoxins or flavodoxins (Blaschkowski et al., Eur. J. Biochem.123:563-569 (1982); Fujii et al., 1977). The Helicobacter pylori FNR,encoded by HP1164 (fqrB), is coupled to the activity ofpyruvate:ferredoxin oxidoreductase (PFOR) resulting in thepyruvate-dependent production of NADPH (St et al. 2007). An analogousenzyme is found in Campylobacter jejuni (St Maurice et al., J.Bacteriol. 189:4764-4773 (2007)). A ferredoxin:NADP+ oxidoreductaseenzyme is encoded in the E. coli genome by fpr (Bianchi et al. 1993).Ferredoxin:NAD+ oxidoreductase utilizes reduced ferredoxin to generateNADH from NAD+. In several organisms, including E. coli, this enzyme isa component of multifunctional dioxygenase enzyme complexes. Theferredoxin:NAD+ oxidoreductase of E. coli, encoded by hcaD, is acomponent of the 3-phenylproppionate dioxygenase system involved ininvolved in aromatic acid utilization (Diaz et al. 1998).NADH:ferredoxin reductase activity was detected in cell extracts ofHydrogenobacter thermophilus, although a gene with this activity has notyet been indicated (Yoon et al. 2006). Additional ferredoxin:NAD(P)+oxidoreductases have been annotated in Clostridium carboxydivorans P7.The NADH-dependent reduced ferredoxin: NADP oxidoreductase of C.kluyveri, encoded by nfnAB, catalyzes the concomitant reduction offerredoxin and NAD+ with two equivalents of NADPH (Wang et al, JBacteriol 192: 5115-5123 (2010)). Finally, the energy-conservingmembrane-associated Rnf-type proteins (Seedorf et al, PNAS 105:2128-2133(2008); and Herrmann, J. Bacteriol 190:784-791 (2008)) provide a meansto generate NADH or NADPH from reduced ferredoxin.

GI Protein GenBank ID Number Organism fqrB NP_207955.1 15645778Helicobacter pylori fqrB YP_001482096.1 157414840 Campylobacter jejuniRPA3954 CAE29395.1 39650872 Rhodopseudomonas palustris Fpr BAH29712.1225320633 Hydrogenobacter thermophilus yumC NP_391091.2 255767736Bacillus subtilis Fpr P28861.4 399486 Escherichia coli hcaD AAC75595.11788892 Escherichia coli LOC100282643 NP_001149023.1 226497434 Zea maysNfnA YP_001393861.1 153953096 Clostridium kluyveri NfnB YP_001393862.1153953097 Clostridium kluyveri CcarbDRAFT_2639 ZP_05392639.1 255525707Clostridium carboxidivorans P7 CcarbDRAFT_2638 ZP_05392638.1 255525706Clostridium carboxidivorans P7 CcarbDRAFT_2636 ZP_05392636.1 255525704Clostridium carboxidivorans P7 CcarbDRAFT_5060 ZP_05395060.1 255528241Clostridium carboxidivorans P7 CcarbDRAFT_2450 ZP_05392450.1 255525514Clostridium carboxidivorans P7 CcarbDRAFT_1084 ZP_05391084.1 255524124Clostridium carboxidivorans P7 RnfC EDK33306.1 146346770 Clostridiumkluyveri RnfD EDK33307.1 146346771 Clostridium kluyveri RnfG EDK33308.1146346772 Clostridium kluyveri RnfE EDK33309.1 146346773 Clostridiumkluyveri RnfA EDK33310.1 146346774 Clostridium kluyveri RnfB EDK33311.1146346775 Clostridium kluyveri CLJU_c11410 (RnfB) ADK14209.1 300434442Clostridium ljungdahlii CLJU_c11400 (RnfA) ADK14208.1 300434441Clostridium ljungdahlii CLJU_c11390 (RnfE) ADK14207.1 300434440Clostridium ljungdahlii CLJU_c11380 (RnfG) ADK14206.1 300434439Clostridium ljungdahlii CLJU_c11370 (RnfD) ADK14205.1 300434438Clostridium ljungdahlii CLJU_c11360 (RnfC) ADK14204.1 300434437Clostridium ljungdahlii MOTH_1518 (NfnA) YP_430370.1 83590361 Moorellathermoacetica MOTH_1517 (NfnB) YP_430369.1 83590360 Moorellathermoacetica CHY_1992 (NfnA) YP_360811.1 78045020 Carboxydothermushydrogenoformans CHY_1993 (NfnB) YP_360812.1 78044266 Carboxydothermushydrogenoformans CLJU_c37220 (NfnAB) YP_003781850.1 300856866Clostridium ljungdahlii

FIG. 1, Step I—Formate Dehydrogenase

Formate dehydrogenase (FDH) catalyzes the reversible transfer ofelectrons from formate to an acceptor. Enzymes with FDH activity utilizevarious electron carriers such as, for example, NADH (EC 1.2.1.2), NADPH(EC 1.2.1.43), quinols (EC 1.1.5.6), cytochromes (EC 1.2.2.3) andhydrogenases (EC 1.1.99.33). FDH enzymes have been characterized fromMorella thermoacetica (Andreesen and Ljungdahl, J Bacteriol 116:867-873(1973); Li et al., J Bacteriol 92:405-412 (1966); Yamamoto et al., JBiol. Chem. 258:1826-1832 (1983). The loci, Moth_2312 is responsible forencoding the alpha subunit of formate dehydrogenase while the betasubunit is encoded by Moth_2314 (Pierce et al., Environ Microbiol(2008)). Another set of genes encoding formate dehydrogenase activitywith a propensity for CO₂ reduction is encoded by Sfum_2703 throughSfum_2706 in Syntrophobacter fumaroxidans (de Bok et al., Eur J.Biochem. 270:2476-2485 (2003)); Reda et al., PNAS 105:10654-10658(2008)). A similar set of genes presumed to carry out the same functionare encoded by CHY_0731, CHY_0732, and CHY_0733 in C. hydrogenoformans(Wu et al., PLoS Genet. 1:e65 (2005)). Formate dehydrogenases are alsofound many additional organisms including C. carboxidivorans P7,Bacillus methanolicus, Burkholderia stabilis, Moorella thermoaceticaATCC 39073, Candida boidinii, Candida methylica, and Saccharomycescerevisiae S288c. The soluble formate dehydrogenase from Ralstoniaeutropha reduces NAD⁺ (fdsG, -B, -A, -C, -D) (Oh and Bowien, 1998)

GI Protein GenBank ID Number Organism Moth_2312 YP_431142 148283121Moorella thermoacetica Moth_2314 YP_431144 83591135 Moorellathermoacetica Sfum_2703 YP_846816.1 116750129 Syntrophobacterfumaroxidans Sfum_2704 YP_846817.1 116750130 Syntrophobacterfumaroxidans Sfum_2705 YP_846818.1 116750131 Syntrophobacterfumaroxidans Sfum_2706 YP_846819.1 116750132 Syntrophobacterfumaroxidans CHY_0731 YP_359585.1 78044572 Carboxydothermushydrogenoformans CHY_0732 YP_359586.1 78044500 Carboxydothermushydrogenoformans CHY_0733 YP_359587.1 78044647 Carboxydothermushydrogenoformans CcarbDRAFT_0901 ZP_05390901.1 255523938 Clostridiumcarboxidivorans P7 CcarbDRAFT_4380 ZP_05394380.1 255527512 Clostridiumcarboxidivorans P7 fdhA, MGA3_06625 EIJ82879.1 387590560 Bacillusmethanolicus MGA3 fdhA, PB1_11719 ZP_10131761.1 387929084 Bacillusmethanolicus PB1 fdhD, MGA3_06630 EIJ82880.1 387590561 Bacillusmethanolicus MGA3 fdhD, PB1_11724 ZP_10131762.1 387929085 Bacillusmethanolicus PB1 fdh ACF35003. 194220249 Burkholderia stabilis FDH1AAC49766.1 2276465 Candida boidinii Fdh CAA57036.1 1181204 Candidamethylica FDH2 P0CF35.1 294956522 Saccharomyces cerevisiae S288c FDH1NP_015033.1 6324964 Saccharomyces cerevisiae S288c

FIG. 1, Step J—Methanol Dehydrogenase

NAD+ dependent methanol dehydrogenase enzymes (EC 1.1.1.244) catalyzethe conversion of methanol and NAD+ to formaldehyde and NADH. An enzymewith this activity was first characterized in Bacillus methanolicus(Heggeset, et al., Applied and Environmental Microbiology,78(15):5170-5181 (2012)). This enzyme is zinc and magnesium dependent,and activity of the enzyme is enhanced by the activating enzyme encodedby act (Kloosterman et al, J Biol Chem 277:34785-92 (2002)). AdditionalNAD(P)+ dependent enzymes can be identified by sequence homology.Methanol dehydrogenase enzymes utilizing different electron acceptorsare also known in the art. Examples include cytochrome dependent enzymessuch as mxaIF of the methylotroph Methylobacterium extorquens (Nunn etal, Nucl Acid Res 16:7722 (1988)). Methanol dehydrogenase enzymes ofmethanotrophs such as Methylococcus capsulatis function in a complexwith methane monooxygenase (MMO) (Myronova et al, Biochem 45:11905-14(2006)). Methanol can also be oxidized to formaldehyde by alcoholoxidase enzymes such as methanol oxidase (EC 1.1.3.13) of Candidaboidinii (Sakai et al, Gene 114: 67-73 (1992)).

GI Protein GenBank ID Number Organism mdh, EIJ77596.1 387585261 Bacillusmethanolicus MGA3_17392 MGA3 mdh2, EIJ83020.1 387590701 Bacillusmethanolicus MGA3_07340 MGA3 mdh3, EIJ80770.1 387588449 Bacillusmethanolicus MGA3_10725 MGA3 act, EIJ83380.1 387591061 Bacillusmethanolicus MGA3_09170 MGA3 mdh, ZP_10132907.1 387930234 Bacillusmethanolicus PB1_17533 PB1 mdh1, ZP_10132325.1 387929648 Bacillusmethanolicus PB1_14569 PB1 mdh2, ZP_10131932.1 387929255 Bacillusmethanolicus PB1_12584 PB1 act, ZP_10132290.1 387929613 Bacillusmethanolicus PB1_14394 PB1 BFZC1_05383 ZP_07048751.1 299535429Lysinibacillus fusiformis BFZC1_20163 ZP_07051637.1 299538354Lysinibacillus fusiformis Bsph_4187 YP_001699778.1 169829620Lysinibacillus sphaericus Bsph_1706 YP_001697432.1 169827274Lysinibacillus sphaericus MCA0299 YP_112833.1 53802410 Methylococcuscapsulatis MCA0782 YP_113284.1 53804880 Methylococcus capsulatis mxaIYP_002965443.1 240140963 Methylobacterium extorquens mxaF YP_002965446.1240140966 Methylobacterium extorquens AOD1 AAA34321.1 170820 Candidaboidinii

FIG. 1, Step K—Spontaneous or Formaldehyde Activating Enzyme

The conversion of formaldehyde and THF to methylenetetrahydrofolate canoccur spontaneously. It is also possible that the rate of this reactioncan be enhanced by a formaldehyde activating enzyme. A formaldehydeactivating enzyme (Fae) has been identified in Methylobacteriumextorquens AM1 which catalyzes the condensation of formaldehyde andtetrahydromethanopterin to methylene tetrahydromethanopterin (Vorholt,et al., J. Bacteriol., 182(23), 6645-6650 (2000)). It is possible that asimilar enzyme exists or can be engineered to catalyze the condensationof formaldehyde and tetrahydrofolate to methylenetetrahydrofolate.Homologs exist in several organisms including Xanthobacter autotrophicusPy2 and Hyphomicrobium denitrificans ATCC 51888.

GI Protein GenBank ID Number Organism MexAM1_META1p1766 Q9FA38.317366061 Methylobacterium extorquens AM1 Xaut_0032 YP_001414948.1154243990 Xanthobacter autotrophicus Py2 Hden_1474 YP_003755607.1300022996 Hyphomicrobium denitrificans ATCC 51888

FIG. 1, Step L—Formaldehyde Dehydrogenase

Oxidation of formaldehyde to formate is catalyzed by formaldehydedehydrogenase. An NAD+ dependent formaldehyde dehydrogenase enzyme isencoded by fdhA of Pseudomonas putida (Ito et al, J Bacteriol 176:2483-2491 (1994)). Additional formaldehyde dehydrogenase enzymes includethe NAD+ and glutathione independent formaldehyde dehydrogenase fromHyphomicrobium zavarzinii (Jerome et al, Appl Microbiol Biotechnol77:779-88 (2007)), the glutathione dependent formaldehyde dehydrogenaseof Pichia pastoris (Sunga et al, Gene 330:39-47 (2004)) and the NAD(P)+dependent formaldehyde dehydrogenase of Methylobacter marinus (Speer etal, FEMS Microbiol Lett, 121(3):349-55 (1994)).

Protein GenBank ID GI Number Organism fdhA P46154.3 1169603 Pseudomonasputida faoA CAC85637.1 19912992 Hyphomicrobium zavarzinii Fld1CCA39112.1 328352714 Pichia pastoris Fdh P47734.2 221222447Methylobacter marinus

In addition to the formaldehyde dehydrogenase enzymes listed above,alternate enzymes and pathways for converting formaldehyde to formateare known in the art. For example, many organisms employglutathione-dependent formaldehyde oxidation pathways, in whichformaldehyde is converted to formate in three steps via theintermediates S-hydroxymethylglutathione and S-formylglutathione(Vorholt et al, J Bacteriol 182:6645-50 (2000)). The enzymes of thispathway are S-(hydroxymethyl)glutathione synthase (EC 4.4.1.22),glutathione-dependent formaldehyde dehydrogenase (EC 1.1.1.284) andS-formylglutathione hydrolase (EC 3.1.2.12).

FIG. 1, Step M—Spontaneous or S-(hydroxymethyl)glutathione Synthase

While conversion of formaldehyde to S-hydroxymethylglutathione can occurspontaneously in the presence of glutathione, it has been shown byGoenrich et al (Goenrich, et al., J Biol Chem 277(5); 3069-72 (2002))that an enzyme from Paracoccus denitrificans can accelerate thisspontaneous condensation reaction. The enzyme catalyzing the conversionof formaldehyde and glutathione was purified and namedglutathione-dependent formaldehyde-activating enzyme (Gfa). The geneencoding it, which was named gfa, is located directly upstream of thegene for glutathione-dependent formaldehyde dehydrogenase, whichcatalyzes the subsequent oxidation of S-hydroxymethylglutathione.Putative proteins with sequence identity to Gfa from P. denitrificansare present also in Rhodobacter sphaeroides, Sinorhizobium meliloti, andMesorhizobium loti.

Protein GenBank ID GI Number Organism Gfa Q51669.3 38257308 Paracoccusdenitrificans Gfa ABP71667.1 145557054 Rhodobacter sphaeroides ATCC17025 Gfa Q92WX6.1 38257348 Sinorhizobium meliloti 1021 Gfa Q98LU4.238257349 Mesorhizobium loti MAFF303099

FIG. 1, Step N—Glutathione-Dependent Formaldehyde Dehydrogenase

Glutathione-dependent formaldehyde dehydrogenase (GS-FDH) belongs to thefamily of class III alcohol dehydrogenases. Glutathione and formaldehydecombine non-enzymatically to form hydroxymethylglutathione, the truesubstrate of the GS-FDH catalyzed reaction. The product,S-formylglutathione, is further metabolized to formic acid.

Protein GenBank ID GI Number Organism frmA YP_488650.1 388476464Escherichia coli K-12 MG1655 SFA1 NP_010113.1 6320033 Saccharomycescerevisiae S288c flhA AAC44551.1 1002865 Paracoccus denitrificans adhIAAB09774.1 986949 Rhodobacter sphaeroides

FIG. 1, Step O—S-Formylglutathione Hydrolase

S-formylglutathione hydrolase is a glutathione thiol esterase found inbacteria, plants and animals. It catalyzes conversion ofS-formylglutathione to formate and glutathione. The fghA gene of P.denitrificans is located in the same operon with gfa and flhA, two genesinvolved in the oxidation of formaldehyde to formate in this organism.In E. coli, FrmB is encoded in an operon with FrmR and FrmA, which areproteins involved in the oxidation of formaldehyde. YeiG of E. coli is apromiscuous serine hydrolase; its highest specific activity is with thesubstrate S-formylglutathione.

Protein GenBank ID GI Number Organism frmB NP_414889.1 16128340Escherichia coli K-12 MG1655 yeiG AAC75215.1 1788477 Escherichia coliK-12 MG1655 fghA AAC44554.1 1002868 Paracoccus denitrificans

4.2 Example II Enhanced Yield of Succinate from Carbohydrates UsingMethanol

Exemplary methanol metabolic pathways for enhancing the availability ofreducing equivalents are provided in FIG. 1.

Succinate production can be achieved in a recombinant organism by thepathway shown in FIG. 2. For example, pathways for the production ofsuccinate from glucose, CO₂, and reducing equivalents (e.g., MeOH) at atheoretical yield of 2.0 mol succinate/mol glucose are provided.Exemplary enzymes for the conversion of glucose to succinate by thisroute include. 2A) a phosphoenolpyruvate (PEP) carboxylase or a PEPcarboxykinase; 2B) a pyruvate carboxylase; 2C) a malate dehydrogenase;2D) a malic enzyme; 2E) a fumarase; and 2F) a fumarate reductase.Succinate production can be carried out by 2A, 2C, 2E and 2F; 2B, 2C, 2Eand 2F; or 2D, 2E and 2F. Oxidative TCA cycle enzymes and enzymes forthe conversion of phosphoenolpyruvate to acetyl-CoA are not required toproduce succinate at the theoretical yield in this example. Note thatseveral other carbohydrates can be converted to phosphoenolpyruvate andsuccinate by this simplified route including xylose, arabinose,galactose, and glycerol.

FIG. 2, Step A—PEP Carboxylase or PEP Carboxykinase

Carboxylation of phosphoenolpyruvate to oxaloacetate is catalyzed byphosphoenolpyruvate carboxylase. Exemplary PEP carboxylase enzymes areencoded by ppc in E. coli (Kai et al., Arch. Biochem. Biophys.414:170-179 (2003), ppcA in Methylobacterium extorquens AMI (Arps etal., J. Bacteriol. 175:3776-3783 (1993), and ppc in Corynebacteriumglutamicum (Eikmanns et al., Mol. Gen. Genet. 218:330-339 (1989).

Protein GenBank ID GI Number Organism Ppc NP_418391 16131794 Escherichiacoli ppcA AAB58883 28572162 Methylobacterium extorquens Ppc ABB5327080973080 Corynebacterium glutamicum

An alternative enzyme for converting phosphoenolpyruvate to oxaloacetateis PEP carboxykinase, which simultaneously forms an ATP whilecarboxylating PEP. In most organisms PEP carboxykinase serves agluconeogenic function and converts oxaloacetate to PEP at the expenseof one ATP. S. cerevisiae is one such organism whose native PEPcarboxykinase, PCK1, serves a gluconeogenic role (Valdes-Hevia et al.,FEBS Lett. 258:313-316 (1989). E. coli is another such organism, as therole of PEP carboxykinase in producing oxaloacetate is believed to beminor when compared to PEP carboxylase, which does not form ATP,possibly due to the higher K_(m) for bicarbonate of PEP carboxykinase(Kim et al., Appl. Environ. Microbiol. 70:1238-1241 (2004)).Nevertheless, activity of the native E. coli PEP carboxykinase from PEPtowards oxaloacetate has been recently demonstrated in ppc mutants of E.coli K-12 (Kwon et al., J. Microbiol. Biotechnol. 16:1448-1452 (2006)).These strains exhibited no growth defects and had increased succinateproduction at high NaHCO₃ concentrations. Alternately, the activity ofthe E. coli enzyme in the oxaloacetate-consuming direction can bereduced by introducing an amino acid substitution at the oxaloacetatebinding site (pck R65Q) (Cotelesage et al., Int. J. Biochem. Cell Biol.39:1204-1210 (2007)). Mutant strains of E. coli can adopt Pck as thedominant CO₂-fixing enzyme following adaptive evolution (Zhang et al.,supra, 2009). In some organisms, particularly rumen bacteria, PEPcarboxykinase is quite efficient in producing oxaloacetate from PEP andgenerating ATP. Examples of PEP carboxykinase genes that have beencloned into E. coli include those from Mannheimia succiniciproducens(Lee et al., Biotechnol. Bioprocess Eng. 7:95-99 (2002)),Anaerobiospirillum succiniciproducens (Laivenieks et al., Appl. Environ.Microbiol. 63:2273-2280 (1997), and Actinobacillus succinogenes (Kim etal. supra). The PEPCK enzyme from Megathyrsus maximus has a low Km forCO₂, a substrate thought to be rate-limiting in the E. coli enzyme (Chenet al., Plant Physiol 128:160-164 (2002); Cotelesage et al., Int. JBiochem. Cell Biol. 39:1204-1210 (2007)). The PEP carboxykinase enzymeof Haemophilus influenza is effective at forming oxaloacetate from PEP.

Protein GenBank ID GI Number Organism PCK1 NP_013023 6322950Saccharomyces cerevisiae Pck NP_417862.1 16131280 Escherichia coli pckAYP_089485.1 52426348 Mannheimia succiniciproducens pckA O09460.1 3122621Anaerobiospirillum succiniciproducens pckA Q6W6X5 75440571Actinobacillus succinogenes AF532733.1:1..1929 AAQ10076.1 33329363Megathyrsus maximus pckA P43923.1 1172573 Haemophilus influenza

FIG. 2, Step B—Pyruvate Carboxylase

Pyruvate carboxylase (EC 6.4.1.1) directly converts pyruvate tooxaloacetate at the cost of one ATP. Pyruvate carboxylase enzymes areencoded by PYC1 (Walker et al., Biochem. Biophys. Res. Commun.176:1210-1217 (1991) and PYC2 (Walker et al., supra) in Saccharomycescerevisiae, and pyc in Mycobacterium smegmatis (Mukhopadhyay andPurwantini, Biochim. Biophys. Acta 1475:191-206 (2000)).

Protein GenBank ID GI Number Organism PYC1 NP_011453 6321376Saccharomyces cerevisiae PYC2 NP_009777 6319695 Saccharomyces cerevisiaePyc YP_890857.1 118470447 Mycobacterium smegmatis

FIG. 2, Step C—Malate Dehydrogenase

Oxaloacetate is converted into malate by malate dehydrogenase (EC1.1.1.37), an enzyme which functions in both the forward and reversedirection. S. cerevisiae possesses three copies of malate dehydrogenase,MDH1 (McAlister-Henn and Thompson, J. Bacteriol. 169:5157-5166 (1987),MDH2 (Minard and McAlister-Henn, Mol. Cell. Biol. 11:370-380 (1991);Gibson and McAlister-Henn, J. Biol. Chem. 278:25628-25636 (2003)), andMDH3 (Steffan and McAlister-Henn, J. Biol. Chem. 267:24708-24715(1992)), which localize to the mitochondrion, cytosol, and peroxisome,respectively. Close homologs to the cytosolic malate dehydrogenase,MDH2, from S. cerevisiae are found in several organisms includingKluyveromyces lactis and Candida tropicalis. E. coli is known to have anactive malate dehydrogenase encoded by mdh.

Protein GenBank ID GI Number Organism MDH1 NP_012838 6322765Saccharomyces cerevisiae MDH2 NP_014515 116006499 Saccharomycescerevisiae MDH3 NP_010205 6320125 Saccharomyces cerevisiae KLLA0E07525pXP_454288.1 50308571 Kluyveromyces lactis NRRL Y-1140 YALI0D16753gXP_502909.1 50550873 Yarrowia lipolytica CTRG_01021 XP_002546239.1255722609 Candida tropicalis MYA-3404 Mdh NP_417703.1 16131126Escherichia coli

FIG. 2, Step D—Malic Enzyme

Malic enzyme can be applied to convert CO₂ and pyruvate to malate at theexpense of one reducing equivalent. Malic enzymes for this purpose caninclude, without limitation, malic enzyme (NAD-dependent) and malicenzyme (NADP-dependent). For example, one of the E. coli malic enzymes(Takeo, J. Biochem. 66:379-387 (1969)) or a similar enzyme with higheractivity can be expressed to enable the conversion of pyruvate and CO₂to malate. By fixing carbon to pyruvate as opposed to PEP, malic enzymeallows the high-energy phosphate bond from PEP to be conserved bypyruvate kinase whereby ATP is generated in the formation of pyruvate orby the phosphotransferase system for glucose transport. Although malicenzyme is typically assumed to operate in the direction of pyruvateformation from malate, overexpression of the NAD-dependent enzyme,encoded by maeA, has been demonstrated to increase succinate productionin E. coli while restoring the lethal Δpfl-ΔldhA phenotype underanaerobic conditions by operating in the carbon-fixing direction (Stolsand Donnelly, Appl. Environ. Microbiol. 63(7) 2695-2701 (1997)). Asimilar observation was made upon overexpressing the malic enzyme fromAscaris suum in E. coli (Stols et al., Appl. Biochem. Biotechnol.63-65(1), 153-158 (1997)). The second E. coli malic enzyme, encoded bymaeB, is NADP-dependent and also decarboxylates oxaloacetate and otheralpha-keto acids (Iwakura et al., J. Biochem. 85(5):1355-65 (1979)).

Protein GenBank ID GI Number Organism maeA NP_415996 90111281Escherichia coli maeB NP_416958 16130388 Escherichia coli NAD-ME P27443126732 Ascaris suum MAE1 NP_012896.1 6322823 Saccharomyces cerevisiaeMAE1 XP_716669.1 68478574 Candida albicans

FIG. 2, Step E—Fumarase

Fumarate hydratase (EC 4.2.1.2) catalyzes the reversible hydration offumarate to malate. The three fumarases of E. coli, encoded by fumA,fumB and fumC, are regulated under different conditions of oxygenavailability. FumB is oxygen sensitive and is active under anaerobicconditions. FumA is active under microanaerobic conditions, and FumC isactive under aerobic growth conditions (Tseng et al., J. Bacteriol.183:461-467 (2001); Woods et al., Biochim. Biophys. Acta 954:14-26(1988); Guest et al., J. Gen. Microbiol. 131:2971-2984 (1985)). S.cerevisiae contains one copy of a fumarase-encoding gene, FUM1, whoseproduct localizes to both the cytosol and mitochondrion (Sass et al., J.Biol. Chem. 278:45109-45116 (2003)). Additional fumarase enzymes arefound in Campylobacter jejuni (Smith et al., Int. J. Biochem. Cell.Biol. 31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch.Biochem. Biophys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi etal., J. Biochem. 89:1923-1931 (1981)). Similar enzymes with highsequence homology include fum1 from Arabidopsis thaliana and fumC fromCorynebacterium glutamicum. The MmcBC fumarase from Pelotomaculumthermopropionicum is another class of fumarase with two subunits(Shimoyama et al., FEMS Microbiol. Lett. 270:207-213 (2007)).

Protein GenBank ID GI Number Organism fumA NP_416129.1 16129570Escherichia coli fumB NP_418546.1 16131948 Escherichia coli fumCNP_416128.1 16129569 Escherichia coli FUM1 NP_015061 6324993Saccharomyces cerevisiae fumC Q8NRN8.1 39931596 Corynebacteriumglutamicum fumC O69294.1 9789756 Campylobacter jejuni fumC P8412775427690 Thermus thermophilus fumH P14408.1 120605 Rattus norvegicusMmcB YP_001211906 147677691 Pelotomaculum thermopropionicum MmcCYP_001211907 147677692 Pelotomaculum thermopropionicum

FIG. 2, Step F—Fumarate Reductase

Fumarate reductase catalyzes the reduction of fumarate to succinate. Thefumarate reductase of E. coli, composed of four subunits encoded byfrdABCD, is membrane-bound and active under anaerobic conditions. Theelectron donor for this reaction is menaquinone and the two protonsproduced in this reaction do not contribute to the proton gradient(Iverson et al., Science 284:1961-1966 (1999)). The yeast genome encodestwo soluble fumarate reductase isozymes encoded by FRDS1 (Enomoto etal., DNA Res. 3:263-267 (1996)) and FRDS2 (Muratsubaki et al., Arch.Biochem. Biophys. 352:175-181 (1998)), which localize to the cytosol andpromitochondrion, respectively, and are used during anaerobic growth onglucose (Arikawa et al., FEMS Microbiol. Lett. 165:111-116 (1998)).

Protein GenBank ID GI Number Organism FRDS1 P32614 418423 Saccharomycescerevisiae FRDS2 NP_012585 6322511 Saccharomyces cerevisiae frdANP_418578.1 16131979 Escherichia coli frdB NP_418577.1 16131978Escherichia coli frdC NP_418576.1 16131977 Escherichia coli frdDNP_418475.1 16131877 Escherichia coli

Succinate dehydrogenase, encoded by sdhCDAB of E. coli, can alsocatalyze the reduction of fumarate to succinate. The reversibility ofboth enzymes is sufficient for SDH and FRD enzymes to complement eachother and support growth if the organism is genetically manipulated toexpress only one or the other. When SDH is expressed under anoxicconditions in the absence of FRD, the SDH complex is able to support alow rate of cell growth by operating as a menaquinol-fumarate reductase(Maklashina et al, J Bacteriol 180:5989-96 (1998)). In another study,Maklashina and coworkers found that the amino acid at position 50 ofSdhA is a key residue for determining directionality of the reaction,and that SDH is a more efficient fumarate reductases when Glu is presentat this position (Maklashina et al, J Biol Chem 281:11357-65 (2006)).

Protein GenBank ID GI Number Organism sdhC NP_415249.1 16128696Escherichia coli sdhD NP_415250.1 16128697 Escherichia coli sdhANP_415251.1 16128698 Escherichia coli sdhB NP_415252.1 16128699Escherichia coli

4.3 Example III Methods of Using Formaldehyde Produced from theOxidation of Methanol in the Formation of Intermediates of CentralMetabolic Pathways for the Formation of Biomass

Provided herein are exemplary pathways, which utilize formaldehydeproduced from the oxidation of methanol (see, e.g., FIG. 1, step J) inthe formation of intermediates of certain central metabolic pathwaysthat can be used for the formation of biomass. Exemplary methanolmetabolic pathways for enhancing the availability of reducingequivalents, as well as the producing formaldehyde from methanol (stepJ), are provided in FIG. 1.

One exemplary pathway that can utilize formaldehyde produced from theoxidation of methanol (e.g., as provided in FIG. 1) is shown in FIG. 3,which involves condensation of formaldehyde and D-ribulose-5-phosphateto form hexylose-6-phosphate (h6p) by hexylose-6-phosphate synthase(FIG. 3, step A). The enzyme can use Mg²⁺ or Mn²⁺ for maximal activity,although other metal ions are useful, and even non-metal-ion-dependentmechanisms are contemplated. H6p is converted into fructose-6-phosphateby 6-phospho-3-hexyloisomerase (FIG. 3, step B).

Another exemplary pathway that involves the detoxification andassimilation of formaldehyde produced from the oxidation of methanol(e.g., as provided in FIG. 1) is shown in FIG. 4 and proceeds throughdihydroxyacetone. Dihydroxyacetone synthase is a special transketolasethat first transfers a glycoaldehyde group from xylulose-5-phosphate toformaldehyde, resulting in the formation of dihydroxyacetone (DHA) andglyceraldehyde-3-phosphate (G3P), which is an intermediate in glycolysis(FIG. 4, step A). The DHA obtained from DHA synthase is then furtherphosphorylated to form DHA phosphate by a DHA kinase (FIG. 4, step B).DHAP can be assimilated into glycolysis and several other pathways.

FIG. 3, Steps A and B—Hexylose-6-Phosphate Synthase (Step A) and6-Phospho-3-hexyloisomerase (Step B)

Both of the hexylose-6-phosphate synthase and6-phospho-3-hexyloisomerase enzymes are found in several organisms,including methanotrops and methylotrophs where they have been purified(Kato et al., 2006, BioSci Biotechnol Biochem. 70(1):10-21. In addition,these enzymes have been reported in heterotrophs such as Bacillussubtilis also where they are reported to be involved in formaldehydedetoxification (Mitsui et al., 2003, AEM 69(10):6128-32, Yasueda et al.,1999. J Bac 181(23):7154-60. Genes for these two enzymes from themethylotrophic bacterium Mycobacterium gastri MB19 have been fused andE. coli strains harboring the hps-phi construct showed more efficientutilization of formaldehyde (Orita et al., 2007, Appl MicrobiolBiotechnol. 76:439-445). In some organisms, these two enzymes naturallyexist as a fused version that is bifunctional.

Exemplary candidate genes for hexylose-6-phopshate synthase are:

Protein GenBank ID GI number Organism Hps AAR39392.1 40074227 Bacillusmethanolicus MGA3 Hps EIJ81375.1 387589055 Bacillus methanolicus PBIRmpA BAA83096.1 5706381 Methylomonas aminofaciens RmpA BAA90546.16899861 Mycobacterium gastri YckG BAA08980.1 1805418 Bacillus subtilis

Exemplary gene candidates for 6-phospho-3-hexyloisomerase are:

Protein GenBank ID GI number Organism Phi AAR39393.1 40074228 Bacillusmethanolicus MGA3 Phi EIJ81376.1 387589056 Bacillus methanolicus PB1 PhiBAA83098.1 5706383 Methylomonas aminofaciens RmpB BAA90545.1 6899860Mycobacterium gastri

Candidates for enzymes where both of these functions have been fusedinto a single open reading frame include the following.

Protein GenBank ID GI number Organism PH1938 NP_143767.1 14591680Pyrococcus horikoshii OT3 PF0220 NP_577949.1 18976592 Pyrococcusfuriosus TK0475 YP_182888.1 57640410 Thermococcus kodakaraensisNP_127388.1 14521911 Pyrococcus abyssi MCA2738 YP_115138.1 53803128Methylococcus capsulatas

FIG. 4, Step A—Dihydroxyacetone synthase

Another exemplary pathway that involves the detoxification andassimilation of formaldehyde produced from the oxidation of methanol(e.g., as provided in FIG. 1) is shown in FIG. 4 and proceeds throughdihydroxyacetone. Dihydroxyacetone synthase is a special transketolasethat first transfers a glycoaldehyde group from xylulose-5-phosphate toformaldehyde, resulting in the formation of dihydroxyacetone (DHA) andglyceraldehyde-3-phosphate (G3P), which is an intermediate in glycolysis(FIG. 4, step A). The DHA obtained from DHA synthase is then furtherphosphorylated to form DHA phosphate by a DHA kinase (FIG. 4, step B).DHAP can be assimilated into glycolysis and several other pathways.

The dihydroxyacetone synthase enzyme in Candida boidinii uses thiaminepyrophosphate and Mg²⁺ as cofactors and is localized in the peroxisome.The enzyme from the methanol-growing carboxydobacterium, Mycobacter sp.strain JC1 DSM 3803, was also found to have DHA synthase and kinaseactivities (Ro et al., 1997, JBac 179(19):6041-7). DHA synthase fromthis organism also has similar cofactor requirements as the enzyme fromC. boidinii. The K_(m)s for formaldehyde and xylulose 5-phosphate werereported to be 1.86 mM and 33.3 microM, respectively. Several othermycobacteria, excluding only Mycobacterium tuberculosis, can usemethanol as the sole source of carbon and energy and are reported to usedihydroxyacetone synthase (Part et al., 2003, JBac 185(1):142-7.

Protein GenBank ID GI number Organism DAS1 AAC83349.1 3978466 Candidaboidinii HPODL_2613 EFW95760.1 320581540 Ogataea parapolymorpha DL-1(Hansenula polymorpha DL-1) AAG12171.2 18497328 Mycobacter sp. strainJC1 DSM 3803

FIG. 4, Step B—Dihydroxyacetone (PHA) Kinase

DHA obtained from DHA synthase is further phosphorylated to form DHAphosphate by a DHA kinase. DHAP can be assimilated into glycolysis andseveral other pathways. Dihydroxyacetone kinase has been purified fromOgataea angusta to homogeneity (Bystrkh, 1983, Biokhimiia,48(10):1611-6). The enzyme, which phosphorylates dihydroxyacetone and,to a lesser degree, glyceraldehyde, is a homodimeric protein of 139 kDa.ATP is the preferred phosphate group donor for the enzyme. When ITP,GTP, CTP and UTP are used, the activity drops to about 30%. In severalorganisms such as Klebsiella pneumoniae and Citrobacter fruendii (Danielet al., 1995, JBac 177(15):4392-40), DHA is formed as a result ofoxidation of glycerol and is converted into DHAP by the kinase DHAkinase of K. pneumoniae has been characterized (Jonathan et al, 1984,JBac 160(1):55-60). It is very specific for DHA, with a K_(m) of 4 μM,and has two apparent K_(m) values for ATP, one at 25 to 35 μM, and theother at 200 to 300 DHA can also be phosphorylated by glycerol kinasesbut the DHA kinase from K. puemoniae is different from glycerol kinasein several respects. While both enzymes can phosphorylatedihydroxyacetone, DHA kinase does not phosphorylate glycerol, neither isit inhibited by fructose-1,6-diphosphate. In Saccharomyces cerevisiae,DHA kinases (I and II) are involved in rescuing the cells from toxiceffects of dihydroxyacetone (Molin et al., 2003, J Biol. Chem. 17;278(3):1415-23).

In Escherichia coli, DHA kinase is composed of the three subunits DhaK,DhaL, and DhaM and it functions similarly to a phosphotransferase system(PTS) in that it utilizes phosphoenolpyruvate as a phosphoryl donor(Gutknecht et al., 2001, EMBO J. 20(10):2480-6). It differs in not beinginvolved in transport. The phosphorylation reaction requires thepresence of the EI and HPr proteins of the PTS system. The DhaM subunitis phosphorylated at multiple sites. DhaK contains the substrate bindingsite (Garcia-Alles et al., 2004, 43(41):13037-45; Siebold et al., 2003,PNAS. 100(14):8188-92). The K_(M) for dihydroxyacetone for the E. colienzyme has been reported to be 6 μM. The K subunit is similar to theN-terminal half of ATP-dependent dihydroxyacetone kinase of Citrobacterfreundii and eukaryotes.

Exemplary DHA kinase gene candidates for this step are:

Protein GenBank ID GI number Organism DAK1 P54838.1 1706391Saccharomyces cerevisiae S288c DAK2 P43550.1 1169289 Saccharomycescerevisiae S288c D186_20916 ZP_16280678.1 421847542 Citrobacter freundiiDAK2 ZP_18488498.1 425085405 Klebsiella pneumoniae DAK AAC27705.13171001 Ogataea angusta DhaK NP_415718.6 162135900 Escherichia coli DhaLNP_415717.1 16129162 Escherichia coli DhaM NP_415716.4 226524708Escherichia coli

4.4 Example III Methods for Handling Anaerobic Cultures

This example describes methods used in handling anaerobic cultures.

A. Anaerobic Chamber and Conditions.

Exemplary anaerobic chambers are available commercially (see, forexample, Vacuum Atmospheres Company, Hawthorne Calif.; MBraun,Newburyport Mass.). Conditions included an O₂ concentration of 1 ppm orless and 1 atm pure N₂. In one example, 3 oxygen scrubbers/catalystregenerators were used, and the chamber included an O₂ electrode (suchas Teledyne; City of Industry CA). Nearly all items and reagents werecycled four times in the airlock of the chamber prior to opening theinner chamber door. Reagents with a volume >5 mL were sparged with pureN₂ prior to introduction into the chamber. Gloves are changed twice/yrand the catalyst containers were regenerated periodically when thechamber displays increasingly sluggish response to changes in oxygenlevels. The chamber's pressure was controlled through one-way valvesactivated by solenoids. This feature allowed setting the chamberpressure at a level higher than the surroundings to allow transfer ofvery small tubes through the purge valve.

The anaerobic chambers achieved levels of O₂ that were consistently verylow and were needed for highly oxygen sensitive anaerobic conditions.However, growth and handling of cells does not usually require suchprecautions. In an alternative anaerobic chamber configuration, platinumor palladium can be used as a catalyst that requires some hydrogen gasin the mix. Instead of using solenoid valves, pressure release can becontrolled by a bubbler. Instead of using instrument-based O₂monitoring, test strips can be used instead.

B. Anaerobic Microbiology.

In particular, serum or media bottles are fitted with thick rubberstoppers and aluminum crimps are employed to seal the bottle. Medium,such as Terrific Broth, is made in a conventional manner and dispensedto an appropriately sized serum bottle. The bottles are sparged withnitrogen for ˜30 mM of moderate bubbling. This removes most of theoxygen from the medium and, after this step, each bottle is capped witha rubber stopper (such as Bellco 20 mm septum stoppers; Bellco,Vineland, N.J.) and crimp-sealed (Bellco 20 mm). Then the bottles ofmedium are autoclaved using a slow (liquid) exhaust cycle. At leastsometimes a needle can be poked through the stopper to provide exhaustduring autoclaving; the needle needs to be removed immediately uponremoval from the autoclave. The sterile medium has the remaining mediumcomponents, for example buffer or antibiotics, added via syringe andneedle. Prior to addition of reducing agents, the bottles areequilibrated for 30-60 minutes with nitrogen (or CO depending upon use).A reducing agent such as a 100×150 mM sodium sulfide, 200 mMcysteine-HCl is added. This is made by weighing the sodium sulfide intoa dry beaker and the cysteine into a serum bottle, bringing both intothe anaerobic chamber, dissolving the sodium sulfide into anaerobicwater, then adding this to the cysteine in the serum bottle. The bottleis stoppered immediately as the sodium sulfide solution generateshydrogen sulfide gas upon contact with the cysteine. When injecting intothe culture, a syringe filter is used to sterilize the solution. Othercomponents are added through syringe needles, such as B12 (10cyanocobalamin), nickel chloride (NiCl₂, 20 microM final concentrationfrom a 40 mM stock made in anaerobic water in the chamber and sterilizedby autoclaving or by using a syringe filter upon injection into theculture), and ferrous ammonium sulfate (final concentration needed is100 μM—made as 100-1000× stock solution in anaerobic water in thechamber and sterilized by autoclaving or by using a syringe filter uponinjection into the culture). To facilitate faster growth under anaerobicconditions, the 1 liter bottles were inoculated with 50 mL of apreculture grown anaerobically. Induction of the pA1-lacO1 promoter inthe vectors was performed by addition of isopropylβ-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mMand was carried out for about 3 hrs.

Large cultures can be grown in larger bottles using continuous gasaddition while bubbling. A rubber stopper with a metal bubbler is placedin the bottle after medium addition and sparged with nitrogen for 30minutes or more prior to setting up the rest of the bottle. Each bottleis put together such that a sterile filter will sterilize the gasbubbled in and the hoses on the bottles are compressible with small Cclamps. Medium and cells are stirred with magnetic stir bars. Once allmedium components and cells are added, the bottles are incubated in anincubator in room air but with continuous nitrogen sparging into thebottles.

4.5 Example IV Strategies for Increased Production of Succinate

This example describes exemplary strategies to increase production ofsuccinate.

Several strain engineering strategies can be implemented to increase theproduction of succinate in an organism and to couple it to growth.

Overexpression of carbon-fixing enzymes such as PEP carboxylase or PEPcarboxykinase (PEPCK), malic enzyme, and pyruvate carboxylase can beused to redirect carbon flux into succinate formation. Cataboliterepression can be removed or reduced by truncating the gene responsiblefor forming cAMP, adenylate cyclase, cyaA (Crasnier et al, Mol. Gen.Genet. 243:409-416 (1994)), and by mutating the catabolite repressorprotein, crp (Eppler and Boos, Mol. Microbiol. 33:1221-1231 (1999);Karimova et al, Res. Microbiol. 155:76-79 (2004); Zhu and Lin, J.Bacteriol. 170:2352-2358 (1988)). Decreasing or eliminating byproductssuch as ethanol, glycerol, acetate, lactate and formate can be used toimprove yields of succinate. In E. coli and other prokaryotes,decreasing or eliminating such byproducts can be effected by deletionsin alcohol dehydrogenase (adhE), lactate dehydrogenase (ldhA), acetatekinase (ackA), pyruvate oxidase (poxB), and pyruvate formate lyase(pflB). The homologue of pflB, pyruvate formate-lyase 2-ketobutyrateformate-lyase (tdcE), can also be deleted in E. coli. Further, deletionof transporters such as the phosphotransferase system (PTS) of glucoseuptake increases the PEP pool in the organism and this has beendemonstrated to improve succinate production in the literature. This canbe accomplished by deletion, mutation or truncation of ptsG, ptsH, ptsIor crr or their combinations (Zhang et al, Proc. Natl. Acad. Sci. USA106(48):20180-20185 (2009), Flores et al, Mol. Microbiol. Biotechnol.13:105-116 (2007); Sanchez et al., Biotechnol. Prog. 21(2):358-365(2005)). This deletion can optionally be accompanied by overexpressionof glucokinase encoded by glk and galactose permease encoded by galP.Similarly, deletion in pyruvate kinase (pykA, pykF) prevents theconversion of PEP to pyruvate and improves succinate production.Further, high concentrations of CO₂ in the fermenters allow the functionof PEPCK and pyruvate carboxylase in the anaplerotic direction, asneeded for succinate production. While exemplified above with specificgenes, it is understood by those skilled in the art that genesperforming the same or similar functions can be genetically modified inthe appropriate host organism to achieve a similar improvement insuccinate production.

Similar strategies to those proposed above can be used for production ofsuccinate in yeasts such as Saccharomyces cerevisiae and Candida. Carbonflux towards succinate can be improved by deleting or tuning downcompeting pathways. Typical fermentation products of yeast includeethanol, glycerol and CO₂. The elimination of these byproducts can beaccomplished by approaches delineated herein. Other potential byproductsinclude lactate, acetate, formate and amino acids.

Ethanol can be formed from pyruvate in two enzymatic steps catalyzed bypyruvate decarboxylase and ethanol dehydrogenase. Saccharomycescerevisiae has three pyruvate decarboxylases (PDC1, PDC5 and PDC6). PDC1is the major isozyme and is strongly expressed in actively fermentingcells. PDC5 also functions during glycolytic fermentation, but isexpressed only in the absence of PDC1 or under thiamine limitingconditions. PDC6 functions during growth on nonfermentable carbonsources. Deleting PDC1 and PDC5 can reduce ethanol productionsignificantly; however these deletions can lead to mutants withincreased PDC6 expression. Deletion of all three eliminates ethanolformation completely but also can cause a growth defect because ofinability of the cells to form sufficient acetyl-CoA for biomassformation. This, however, can be overcome by evolving cells in thepresence of reducing amounts of C2 carbon source (ethanol or acetate)(van Maris et al, AEM 69:2094-9 (2003)). It has also been reported thatdeletion of the positive regulator PDC2 of pyruvate decarboxylases PDC1and PDC5, reduced ethanol formation to ˜10% of that made by wild-type(Hohmann et al, Mol Gen Genet. 241:657-66 (1993)). Pyruvatedecarboxylase (PDC) is a key enzyme in alcoholic fermentation,catalyzing the decarboxylation of pyruvate to acetaldehyde. The PDC1enzyme from Saccharomyces cerevisiae has been extensively studied(Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001); Li etal., Biochemistry. 38:10004-10012 (1999); ter Schure et al., Appl.Environ. Microbiol. 64:1303-1307 (1998)). Other well-characterized PDCenzymes are found in Zymomonas mobilus (Siegert et al., Protein Eng DesSel 18:345-357 (2005)), Acetobacter pasteurians (Chandra et al.,176:443-451 (2001)) and Kluyveromyces lactis (Krieger et al.,269:3256-3263 (2002)). The PDC1 and PDC5 enzymes of Saccharomycescerevisiae are subject to positive transcriptional regulation by PDC2(Hohmann et al, Mol Gen Genet. 241:657-66 (1993)). Pyruvatedecarboxylase activity is also possessed by a protein encoded byCTRG_03826 (GI:255729208) in Candida tropicalis, PDC1 (GI number:1226007) in Kluyveromyces lactis, YALI0D10131g (GI:50550349) in Yarrowialipolytica, PAS_chr3_0188 (GI:254570575) in Pichia pastoris, pyruvatedecarboxylase (GI: GI:159883897) in Schizosaccharomyces pombe,ANI_1_1024084 (GI:145241548), ANI_1_796114 (GI:317034487), ANI_1_936024(GI:317026934) and ANI_1_2276014 (GI:317025935) in Aspergillus niger.

GI Protein GenBank ID Number Organism Pdc P06672.1 118391 Zymomonasmobilis pdc1 P06169 30923172 Saccharomyces cerevisiae Pdc2 NP_010366.16320286 Saccharomyces cerevisiae Pdc5 NP_013235.1 6323163 Saccharomycescerevisiae CTRG_03826 XP_002549529 255729208 Candida tropicalis,CU329670.1: CAA90807 159883897 Schizosaccharomyces 585597.587312 pombeYALI0D10131g XP_502647 50550349 Yarrowia lipolytica PAS_chr3_0188XP_002492397 254570575 Pichia pastoris Pdc Q8L388 20385191 Acetobacterpasteurians pdc1 Q12629 52788279 Kluyveromyces lactis ANI_1_1024084XP_001393420 145241548 Aspergillus niger ANI_1_796114 XP_001399817317026934 Aspergillus niger ANI_1_936024 XP_001396467 317034487Aspergillus niger ANI_1_2276014 XP_001388598 317025935 Aspergillus niger

Ethanol dehydrogenases that convert acetaldehyde into ethanol and/orother short chain alcohol dehydrogenases can be deleted or attenuated toprovide carbon and reducing equivalents for the succinate pathway. Todate, seven alcohol dehydrogenases, ADHI-ADHVII, have been reported inS. cerevisiae (de Smidt et al, FEMS Yeast Res 8:967-78 (2008)). ADH1(GI:1419926) is the key enzyme responsible for reducing acetaldehyde toethanol in the cytosol under anaerobic conditions. It has been reportedthat a yeast strain deficient in ADH1 cannot grow anaerobically becausean active respiratory chain is the only alternative path to regenerateNADH and lead to a net gain of ATP (Drewke et al, J Bacteriol172:3909-17 (1990)). This enzyme is an ideal candidate fordownregulation to limit ethanol production. ADH2 is severely repressedin the presence of glucose. In K. lactis, two NAD-dependent cytosolicalcohol dehydrogenases have been identified and characterized. Thesegenes also show activity for other aliphatic alcohols. The genes ADH1(GI:113358) and ADHII (GI:51704293) are preferentially expressed inglucose-grown cells (Bozzi et al, Biochim Biophys Acta 1339:133-142(1997)). Cytosolic alcohol dehydrogenases are encoded by ADH1(GI:608690) in C. albicans, ADH1 (GI:3810864) in S. pombe, ADH1(GI:5802617) in Y. lipolytica, ADH1 (GI:2114038) and ADHII (GI:2143328)in Pichia stipitis or Scheffersomyces stipitis (Passoth et al, Yeast14:1311-23 (1998)). Candidate alcohol dehydrogenases are shown the tablebelow.

Protein GenBank ID GI number Organism SADH BAA24528.1 2815409 Candidaparapsilosis ADH1 NP_014555.1 6324486 Saccharomyces cerevisiae s288cADH2 NP_014032.1 6323961 Saccharomyces cerevisiae s288c ADH3 NP_013800.16323729 Saccharomyces cerevisiae s288c ADH4 NP_011258.2 269970305Saccharomyces cerevisiae s288c ADH5 (SFA1) NP_010113.1 6320033Saccharomyces cerevisiae s288c ADH6 NP_014051.1 6323980 Saccharomycescerevisiae s288c ADH7 NP_010030.1 6319949 Saccharomyces cerevisiae s288cadhP CAA44614.1 2810 Kluyveromyces lactis ADH1 P20369.1 113358Kluyveromyces lactis ADH2 CAA45739.1 2833 Kluyveromyces lactis ADH3P49384.2 51704294 Kluyveromyces lactis ADH1 CAA57342.1 608690 Candidaalbicans ADH2 CAA21988.1 3859714 Candida albicans SAD XP_712899.168486457 Candida albicans ADH1 CAA21782.1 3810864 Schizosaccharomycespombe ADH1 AAD51737.1 5802617 Yarrowia lipolytica ADH2 AAD51738.15802619 Yarrowia lipolytica ADH3 AAD51739.1 5802621 Yarrowia lipolyticaAlcB AAX53105.1 61696864 Aspergillus niger ANI_1_282024 XP_001399347.1145231748 Aspergillus niger ANI_1_126164 XP_001398574.2 317037131Aspergillus niger ANI_1_1756104 XP_001395505.2 317033815 Aspergillusniger ADH2 CAA73827.1 2143328 Scheffersomyces stipitis

Attenuation or deletion of one or more glycerol-3-phosphatase orglycerol-3-phosphate (G3P) dehydrogenase enzymes can eliminate or reducethe formation of glycerol, and thereby conserve carbon and reducingequivalents for production of succinate.

G3P phosphatase catalyzes the hydrolysis of G3P to glycerol. Enzymeswith this activity include the glycerol-1-phosphatase (EC 3.1.3.21)enzymes of Saccharomyces cerevisiae (GPP1 and GPP2), Candida albicansand Dunaleilla parva (Popp et al, Biotechnol Bioeng 100:497-505 (2008);Fan et al, FEMS Microbial Lett 245:107-16 (2005)). The D. parva gene hasnot been identified to date. These and additional G3P phosphataseenzymes are shown in the table below.

Protein GenBank ID GI Number Organism GPP1 DAA08494.1 285812595Saccharomyces cerevisiae GPP2 NP_010984.1 6320905 Saccharomycescerevisiae GPP1 XP_717809.1 68476319 Candida albicans KLLA0C08217gXP_452565.1 50305213 Kluyveromyces lactis KLLA0C11143g XP_452697.150305475 Kluyveromyces lactis ANI_1_380074 XP_001392369.1 145239445Aspergillus niger ANI_1_444054 XP_001390913.2 317029125 Aspergillusniger

S. cerevisiae has three G3P dehydrogenase enzymes encoded by GPD1 andGDP2 in the cytosol and GUT2 in the mitochondrion. GPD2 is known toencode the enzyme responsible for the majority of the glycerol formationand is responsible for maintaining the redox balance under anaerobicconditions. GPD1 is primarily responsible for adaptation of S.cerevisiae to osmotic stress (Bakker et al., FEMS Microbiol Rev 24:15-37(2001)). Attenuation of GPD1, GPD2 and/or GUT2 will reduce glycerolformation. GPD1 and GUT2 encode G3P dehydrogenases in Yarrowialipolytica (Beopoulos et al, AEM 74:7779-89 (2008)). GPD1 and GPD2encode for G3P dehydrogenases in S. pombe. Similarly, G3P dehydrogenaseis encoded by CTRG_02011 in Candida tropicalis and a gene represented byGI:20522022 in Candida albicans.

Protein GenBank ID GI number Organism GPD1 CAA98582.1 1430995Saccharomyces cerevisiae GPD2 NP_014582.1 6324513 Saccharomycescerevisiae GUT2 NP_012111.1 6322036 Saccharomyces cerevisiae GPD1CAA22119.1 6066826 Yarrowia lipolytica GUT2 CAG83113.1 49646728 Yarrowialipolytica GPD1 CAA22119.1 3873542 Schizosaccharomyces pombe GPD2CAA91239.1 1039342 Schizosaccharomyces pombe ANI_1_786014 XP_001389035.2317025419 Aspergillus niger ANI_1_1768134 XP_001397265.1 145251503Aspergillus niger KLLA0C04004g XP_452375.1 50304839 Kluyveromyces lactisCTRG_02011 XP_002547704.1 255725550 Candida tropicalis GPD1 XP_714362.168483412 Candida albicans GPD2 XP_713824.1 68484586 Candida albicans

Enzymes that form acid byproducts such as acetate, formate and lactatecan also be tuned down or deleted. Such enzymes include acetate kinase,phosphotransacetylase and pyruvate oxidase.

An exemplary acetate kinase is the E. coli acetate kinase, encoded byackA (Skarstedt and Silverstein J. Biol. Chem. 251:6775-6783 (1976)).Homologs exist in several other organisms including Salmonella entericaand Chlamydomonas reinhardtii. Information related to these proteins andgenes is shown below:

Protein GenBank ID GI number Organism AckA NP_416799.1 16130231Escherichia coli AckA NP_461279.1 16765664 Salmonella enterica subsp.enterica serovar Typhimurium str. LT2 ACK1 XP_001694505.1 159472745Chlamydomonas reinhardtii ACK2 XP_001691682.1 159466992 Chlamydomonasreinhardtii

An exemplary phosphate-transferring acyltransferase isphosphotransacetylase, encoded by pta. The pta gene from E. coli encodesan enzyme that can convert acetyl-CoA into acetyl-phosphate, and viceversa (Suzuki, T. Biochim. Biophys. Acta 191:559-569 (1969)). Thisenzyme can also utilize propionyl-CoA instead of acetyl-CoA formingpropionate in the process (Hesslinger et al. Mol. Microbiol 27:477-492(1998)). Homologs exist in several other organisms including Salmonellaenterica and Chlamydomonas reinhardtii.

Protein GenBank ID GI number Organism Pta NP_416800.1 16130232Escherichia coli Pta NP_461280.1 16765665 Salmonella enterica subsp.enterica serovar Typhimurium str. LT2 PAT2 XP_001694504.1 159472743Chlamydomonas reinhardtii PAT1 XP_001691787.1 159467202 Chlamydomonasreinhardtii

Pyruvate oxidase (acetate-forming) or pyruvate:quinone oxidoreductase(PQO) catalyzes the oxidative decarboxylation of pyruvate into acetate,using ubiquione (EC 1.2.5.1) or quinone (EC 1.2.2.1) as an electronacceptor. The E. coli enzyme, PoxB, is localized on the inner membrane(Abdel-Hamid et al., Microbiol 147:1483-98 (2001)). The enzyme hasthiamin pyrophosphate and flavin adenine dinucleotide (FAD) cofactors(Koland and Gennis, Biochemistry 21:4438-4442 (1982)); O'Brien et al.,Biochemistry 16:3105-3109 (1977); O'Brien and Gennis, J. Biol. Chem.255:3302-3307 (1980)). PoxB has similarity to pyruvate decarboxylase ofS. cerevisiae and Zymomonas mobilis. The pqo transcript ofCorynebacterium glutamicum encodes a quinone-dependent andacetate-forming pyruvate oxidoreductase (Schreiner et al., J Bacteriol188:1341-50 (2006)). Similar enzymes can be inferred by sequencehomology.

Protein GenBank ID GI Number Organism poxB NP_415392.1 16128839Escherichia coli Pqo YP_226851.1 62391449 Corynebacterium glutamicumpoxB YP_309835.1 74311416 Shigella sonnei poxB ZP_03065403.1 194433121Shigella dysenteriae

Deletion or attenuation of pyruvate formate lyase could limit formationof formate. Pyruvate formate-lyase (PFL, EC 2.3.1.54), encoded by pflBin E. coli, can convert pyruvate into acetyl-CoA and formate. Theactivity of PFL can be enhanced by an activating enzyme encoded by pflA(Knappe et al., Proc. Natl. Acad. Sci U.S.A 81:1332-1335 (1984); Wong etal., Biochemistry 32:14102-14110 (1993)). Keto-acid formate-lyase (EC2.3.1.-), also known as 2-ketobutyrate formate-lyase (KFL) and pyruvateformate-lyase 4, is the gene product of tdcE in E. coli. This enzymecatalyzes the conversion of 2-ketobutyrate to propionyl-CoA and formateduring anaerobic threonine degradation, and can also substitute forpyruvate formate-lyase in anaerobic catabolism (Simanshu et al., JBiosci. 32:1195-1206 (2007)). The enzyme is oxygen-sensitive and, likePflB, can require post-translational modification by PFL-AE to activatea glycyl radical in the active site (Hesslinger et al., Mol. Microbiol27:477-492 (1998)). A pyruvate formate-lyase from Archaeglubus fulgidusencoded by pflD has been cloned, expressed in E. coli and characterized(Lehtio et al., Protein Eng Des Sel 17:545-552 (2004)). The crystalstructures of the A. fulgidus and E. coli enzymes have been resolved(Lehtio et al., J. Mol. Biol. 357:221-235 (2006); Leppanen et al.,Structure. 7:733-744 (1999)). Additional PFL and PFL-AE candidates arefound in Lactococcus lactis (Melchiorsen et al., Appl MicrobiolBiotechnol 58:338-344 (2002)), and Streptococcus mutans (Takahashi-Abbeet al., Oral. Microbiol Immunol. 18:293-297 (2003)), Chlamydomonasreinhardtii (Hemschemeier et al., Eukaryot. Cell 7:518-526 (2008b);Atteia et al., J. Biol. Chem. 281:9909-9918 (2006)) and Clostridiumpasteurianum (Weidner et al., J. Bacteriol. 178:2440-2444 (1996)).

Protein GenBank ID GI Number Organism pflB NP_415423 16128870Escherichia coli pflA NP_415422.1 16128869 Escherichia coli tdcEAAT48170.1 48994926 Escherichia coli pflD NP_070278.1 11499044Archaeglubus fulgidus Pfl CAA03993 2407931 Lactococcus lactis PflBAA09085 1129082 Streptococcus mutans PFL1 XP_001689719.1 159462978Chlamydomonas reinhardtii pflA1 XP_001700657.1 159485246 Chlam domonasreinhardtii Pfl Q46266.1 2500058 Clostridium pasteurianum Act CAA63749.11072362 Clostridium pasteurianum

Alcohol dehydrogenases that convert pyruvate to lactate are alsocandidates for deletion or attenuation. Lactate dehydrogenase enzymesinclude ldhA of E. coli and ldh from Ralstonia eutropha (Steinbuchel andSchlegel, Eur. J. Biochem. 130:329-334 (1983)). Other alcoholdehydrogenases listed above may also exhibit LDH activity.

Protein GenBank ID GI number Organism ldhA NP_415898.1 16129341Escherichia coli Ldh YP_725182.1 113866693 Ralstonia eutropha

Tuning down activity of the mitochondrial pyruvate dehydrogenase complexwill limit flux into the mitochondrial TCA cycle. Under anaerobicconditions and in conditions where glucose concentrations are high inthe medium, the capacity of this mitochondrial enzyme is very limitedand there is no significant flux through it. However, in someembodiments, this enzyme can be disrupted or attenuated to increasesuccinate production. Exemplary pyruvate dehydrogenase genes includePDB1, PDA1, LAT1 and LPD1.

The pyruvate dehydrogenase (PDH) complex catalyzes the conversion ofpyruvate to acetyl-CoA. The E. coli PDH complex is encoded by the genesaceEF and lpdA. Enzyme engineering efforts have improved the E. coli PDHenzyme activity under anaerobic conditions (Kim et al., J. Bacteriol.190:3851-3858 (2008); Kim et al., Appl Environ. Microbiol. 73:1766-1771(2007); Zhou et al., Biotechnol Lett. 30:335-342 (2008)). In contrast tothe E. coli PDH, the B. subtilis complex is active and required forgrowth under anaerobic conditions (Nakano et al., 179:6749-6755 (1997)).The Klebsiella pneumoniae PDH, characterized during growth on glycerol,is also active under anaerobic conditions (Menzel et al., 56:135-142(1997)). Crystal structures of the enzyme complex from bovine kidney(Zhou et al., 98:14802-14807 (2001)) and the E2 catalytic domain fromAzotobacter vinelandii are available (Mattevi et al., Science.255:1544-1550 (1992)). Some mammalian PDH enzymes complexes can react onalternate substrates such as 2-oxobutanoate. Comparative kinetics ofRattus norvegicus PDH and BCKAD indicate that BCKAD has higher activityon 2-oxobutanoate as a substrate (Paxton et al., Biochem. 1 234:295-303(1986)). The S. cerevisiae PDH complex canconsist of an E2 (LAT1) corethat binds E1 (PDA), PDB1), E3 (LPD1), and Protein X (PDX1) components(Pronk et al., Yeast 12:1607-1633 (1996)). The PDH complex of S.cerevisiae is regulated by phosphorylation of E1 involving PKP1 (PDHkinase I), PTC5 (PDH phosphatase 1), PKP2 and PTC6. Modification ofthese regulators may also enhance PDH activity. Coexpression of lipoylligase (Lp1A of E. coli and AIM22 in S. cerevisiae) with PDH in thecytosol may be necessary for activating the PDH enzyme complex.Increasing the supply of cytosolic lipoate, either by modifying ametabolic pathway or media supplementation with lipoate, may alsoimprove PDH activity.

Gene Accession No. GI Number Organism aceE NP_414656.1 16128107Escherichia coli aceF NP_414657.1 16128108 Escherichia coli LpdNP_414658.1 16128109 Escherichia coli lplA NP_418803.1 16132203Escherichia coli pdhA P21881.1 3123238 Bacillus subtilis pdhB P21882.1129068 Bacillus subtilis pdhC P21883.2 129054 Bacillus subtilis pdhDP21880.1 118672 Bacillus subtilis aceE YP_001333808.1 152968699Klebsiella pneumoniae aceF YP_001333809.1 152968700 Klebsiellapneumoniae lpdA YP_001333810.1 152968701 Klebsiella pneumoniae Pdha1NP_001004072.2 124430510 Rattus norvegicus Pdha2 NP_446446.1 16758900Rattus norvegicus Dlat NP_112287.1 78365255 Rattus norvegicus DldNP_955417.1 40786469 Rattus norvegicus LAT1 NP_014328 6324258Saccharomyces cerevisiae PDA1 NP_011105 37362644 Saccharomycescerevisiae PDB1 NP_009780 6319698 Saccharomyces cerevisiae LPD1NP_116635 14318501 Saccharomyces cerevisiae PDX1 NP_011709 6321632Saccharomyces cerevisiae AIM22 NP_012489.2 83578101 Saccharomycescerevisiae

Another strategy for reducing flux into the TCA cycle is to limittransport of pyruvate into the mitochondria by tuning down or deletingthe mitochondrial pyruvate carrier. Transport of pyruvate into themitochondria in S. cerevisiae is catalyzed by a heterocomplex encoded byMPC1 and MPC2 (Herzig et al, Science 337:93-6 (2012); Bricker et al,Science 337:96-100 (2012)). S. cerevisiae encodes five other putativemonocarboxylate transporters (MCH1-5), several of which may be localizedto the mitochondrial membrane (Makuc et al, Yeast 18:1131-43 (2001)).NDT1 is another putative pyruvate transporter, although the role of thisprotein is disputed in the literature (Todisco et al, J Biol Chem20:1524-31 (2006)). Exemplary pyruvate and monocarboxylate transportersare shown in the table below:

Protein GenBank ID GI number Organism MPC1 NP_011435.1 6321358Saccharomyces cerevisiae MPC2 NP_012032.1 6321956 Saccharomycescerevisiae MPC1 XP_504811.1 50554805 Yarrowia lipolytica MPC2XP_501390.1 50547841 Yarrowia lipolytica MPC1 XP_719951.1 68471816Candida albicans MPC2 XP_716190.1 68479656 Candida albicans MCH1NP_010229.1 6320149 Saccharomyces cerevisiae MCH2 NP_012701.2 330443640Saccharomyces cerevisiae MCH3 NP_014274.1 6324204 Saccharomycescerevisiae MCH5 NP_014951.2 330443742 Saccharomyces cerevisiae NDT1NP_012260.1 6322185 Saccharomyces cerevisiae ANI_1_1592184XP_001401484.2 317038471 Aspergillus niger CaJ7_0216 XP_888808.177022728 Candida albicans YALI0E16478g XP_504023.1 50553226 Yarrowialipolytica KLLA0D14036g XP_453688.1 50307419 Kluyveromyces lactis

One exemplary method to provide an increased number of reducingequivalents, such as NAD(P)H, for enabling the formation of succinate isto constrain the use of such reducing equivalents during respiration.Respiration can be limited by: reducing the availability of oxygen,attenuating NADH dehydrogenases and/or cytochrome oxidase activity,attenuating G3P dehydrogenase, and/or providing excess glucose toCrabtree positive organisms.

Restricting oxygen availability by culturing the non-naturally occurringeukaryotic organisms in a fermenter is one approach for limitingrespiration and thereby increasing the ratio of NAD(P)H to NAD(P). Theratio of NAD(P)H/NAD(P) increases as culture conditions get moreanaerobic, with completely anaerobic conditions providing the highestratios of the reduced cofactors to the oxidized ones. For example, ithas been reported that the ratio of NADH/NAD=0.02 in aerobic conditionsand 0.75 in anaerobic conditions in E. coli (de Graes et al, J Bacteriol181:2351-57 (1999)).

Respiration can also be limited by reducing expression or activity ofNADH dehydrogenases and/or cytochrome oxidases in the cell under aerobicconditions. In this case, respiration will be limited by the capacity ofthe electron transport chain. Such an approach has been used to enableanaerobic metabolism of E. coli under completely aerobic conditions(Portnoy et al, AEM 74:7561-9 (2008)). S. cerevisiae can oxidizecytosolic NADH directly using external NADH dehydrogenases, encoded byNDE1 and NDE2. One such NADH dehydrogenase in Yarrowia lipolytica isencoded by NDH2 (Kerscher et al, J Cell Sci 112:2347-54 (1999)). Theseand other NADH dehydrogenase enzymes are listed in the table below.

Protein GenBank ID GI number Organism NDE1 NP_013865.1 6323794Saccharomyces cerevisiae s288c NDE2 NP_010198.1 6320118 Saccharomycescerevisiae s288c NDH2 AJ006852.1 3718004 Yarrowia lipolyticaANI_1_610074 XP_001392541.2 317030427 Aspergillus niger ANI_1_2462094XP_001394893.2 317033119 Aspergillus niger KLLA0E21891g XP_454942.150309857 Kluyveromyces lactis KLLA0C06336g XP_452480.1 50305045Kluyveromyces lactis NDE1 XP_720034.1 68471982 Candida albicans NDE2XP_717986.1 68475826 Candida albicans

Cytochrome oxidases of Saccharomyces cerevisiae include the COX geneproducts. COX1-3 are the three core subunits encoded by themitochondrial genome, whereas COX4-13 are encoded by nuclear genes.Attenuation or deletion of any of the cytochrome genes results in adecrease or block in respiratory growth (Hermann and Funes, Gene354:43-52 (2005)). Cytochrome oxidase genes in other organisms can beinferred by sequence homology.

Protein GenBank ID GI number Organism COX1 CAA09824.1 4160366Saccharomyces cerevisiae s288c COX2 CAA09845.1 4160387 Saccharomycescerevisiae s288c COX3 CAA09846.1 4160389 Saccharomyces cerevisiae s288cCOX4 NP_011328.1 6321251 Saccharomyces cerevisiae s288c COX5ANP_014346.1 6324276 Saccharomyces cerevisiae s288c COX5B NP_012155.16322080 Saccharomyces cerevisiae s288c COX6 NP_011918.1 6321842Saccharomyces cerevisiae s288c COX7 NP_013983.1 6323912 Saccharomycescerevisiae s288c COX8 NP_013499.1 6323427 Saccharomyces cerevisiae s288cCOX9 NP_010216.1 6320136 Saccharomyces cerevisiae s288c COX12NP_013139.1 6323067 Saccharomyces cerevisiae s288c COX13 NP_011324.16321247 Saccharomyces cerevisiae s288c

Cytosolic NADH can also be oxidized by the respiratory chain via the G3Pdehydrogenase shuttle, consisting of cytosolic NADH-linked G3Pdehydrogenase and a membrane-bound G3P:ubiquinone oxidoreductase. Thedeletion or attenuation of G3P dehydrogenase enzymes will also preventthe oxidation of NADH for respiration. Enzyme candidates encoding theseenzymes were described above.

Additionally, in Crabtree positive organisms, fermentative metabolismcan be achieved in the presence of excess of glucose. For example, S.cerevisiae makes ethanol even under aerobic conditions. The formation ofethanol and glycerol can be reduced/eliminated and replaced by theproduction of succinate in a Crabtree positive organism by feedingexcess glucose to the Crabtree positive organism. In another aspectprovided herein is a method for producing succinate, comprisingculturing a non-naturally occurring eukaryotic organism under conditionsand for a sufficient period of time to produce succinate, wherein theeukaryotic organism is a Crabtree positive organism that comprises atleast one nucleic acid encoding a succinate pathway enzyme and whereineukaryotic organism is in a culture medium comprising excess glucose.

Reduced activity of pyruvate kinase encoded by pyk1 and pyk2 can also beused to increase the pool of PEP for succinate production. Pyruvatekinase, also known as phosphoenolpyruvate synthase (EC 2.7.9.2),converts pyruvate and ATP to PEP and AMP. This enzyme is encoded by thePYK1 (Burke et al., J. Biol. Chem. 258:2193-2201 (1983)) and PYK2 (Boleset al., J. Bacteriol. 179:2987-2993 (1997)) genes in S. cerevisiae. InE. coli, this activity is catalyzed by the gene products of pykF andpykA. Selected homologs of the S. cerevisiae enzymes are also shown inthe table below.

Protein GenBank ID GI Number Organism PYK1 NP_009362 6319279Saccharomyces cerevisiae PYK2 NP_014992 6324923 Saccharomyces cerevisiaepykF NP_416191.1 16129632 Escherichia coli pykA NP_416368.1 16129807Escherichia coli KLLA0F23397g XP_4456122.1 50312181 Kluyveromyces lactisCaO19.3575 XP_714934.1 68482353 Candida albicans CaO19.11059 XP_714997.168482226 Candida albicans YALI0F09185p XP_505195 210075987 Yarrowialipolytica ANI_1_1126064 XP_001391973 145238652 Aspergillus niger

4.6 Example V Increased Production of Succinate in Microbial Organisms

This example describes engineering of microbial organisms for increasedproduction of succinate.

In E. coli, the relevant genes are expressed in a synthetic operonbehind an inducible promoter on a medium- or high-copy plasmid; forexample the PBAD promoter which is induced by arabinose, on a plasmid ofthe pBAD series (Guzman et al., J Bacteriol. 177:4121-4130 (1995)). InS. cerevisiae, genes are integrated into the chromosome behind the PDC1promoter, replacing the native pyruvate carboxylase gene. It has beenreported that this results in higher expression of foreign genes thanfrom a plasmid (Ishida et al., Appl. Environ. Microbiol. 71:1964-1970(2005)), and will also ensure expression during anaerobic conditions.

Cells containing the relevant constructs are grown in suitable growthmedia, for example, minimal media containing glucose. The addition ofarabinose can be included in the case of E. coli containing genesexpressed under the PBAD promoter. Periodic samples are taken for bothgene expression and enzyme activity analysis. Enzyme activity assays areperformed on crude cell extracts using procedures well known in the art.Alternatively, assays based on the oxidation of NAD(P)H, which isproduced in all dehydrogenase reaction steps and detectable byspectrophotometry can be utilized. In addition, antibodies can be usedto detect the level of particular enzymes. In lieu of or in addition toenzyme activity measurements, RNA can be isolated from parallel samplesand transcript of the gene of interest measured by reverse transcriptasePCR. Any constructs lacking detectable transcript expression arereanalyzed to ensure the encoding nucleic acids are harbored in anexpressible form. Where transcripts are detected, this result indicateseither a lack of translation or production of inactive enzyme. A varietyof methods well known in the art can additionally be employed, such ascodon optimization, engineering a strong ribosome binding site, use of agene from a different species, and prevention of N-glycosylation (forexpression of bacterial enzymes in yeast) by conversion of Asn residuesto Asp. Once all required enzyme activities are detected, the next stepis to measure the production of succinate in vivo. Triplicate shakeflask cultures are grown aerobically, anaerobically or microaerobically,depending on the conditions required, and periodic samples taken.Organic acids present in the culture supernatants are analyzed by HPLCusing the Aminex AH-87X column. The elution time of succinate will bedetermined using a standard purchased from a chemical supplier.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties, includingGenBank and GI number publications, are hereby incorporated by referencein this application in order to more fully describe the state of the artto which this invention pertains. Although the invention has beendescribed with reference to the examples and embodiments provided above,it should be understood that various modifications can be made withoutdeparting from the spirit of the invention.

What is claimed is:
 1. A non-naturally occurring microbial organismcomprising: (a) a methanol metabolic pathway, wherein said non-naturallyoccurring microbial organism comprises at least one exogenous nucleicacid encoding a methanol metabolic pathway enzyme expressed in asufficient amount to enhance the availability of reducing equivalents inthe presence of methanol or metabolize methanol as a carbon source forbiosynthesis of succinate, wherein said methanol metabolic pathwaycomprises: (i) a methanol methyltransferase and amethylenetetrahydrofolate reductase; (ii) a methanol dehydrogenase; or(iii) a methanol dehydrogenase and a formaldehyde activating enzyme; and(b) a succinate pathway comprising exogenous nucleic acids wherein atleast one of said exogenous nucleic acids is a heterologous nucleic acidencoding a succinate pathway enzyme expressed in a sufficient amount toproduce succinate, said succinate pathway comprising: (i) aphosphoenolpyruvate (PEP) carboxylase or a PEP carboxykinase, a malatedehydrogenase, a fumarase, and a fumarate reductase; (ii) a pyruvatecarboxylase, a malate dehydrogenase, a fumarase, and a fumaratereductase; or (iii) a malic enzyme, a fumarase, and a fumaratereductase.
 2. The non-naturally occurring microbial organism of claim 1,wherein the non-naturally occurring microbial organism comprises two,three, or four heterologous nucleic acids, each encoding a succinatepathway enzyme.
 3. The non-naturally occurring microbial organism ofclaim 1, wherein the methanol metabolic pathway comprises (i) a methanolmethyltransferase, a methylenetetrahydrofolate reductase, amethylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolatecyclohydrolase, and a formyltetrahydrofolate deformylase; (ii) amethanol methyltransferase, a methylenetetrahydrofolate reductase, amethylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolatecyclohydrolase and a formyltetrahydrofolate synthetase; (iii) a methanoldehydrogenase, a methylenetetrahydrofolate dehydrogenase, amethenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolatedeformylase; (iv) a methanol dehydrogenase, a methylenetetrahydrofolatedehydrogenase, a methenyltetrahydrofolate cyclohydrolase and aformyltetrahydrofolate synthetase; (v) a methanol dehydrogenase and aformaldehyde dehydrogenase; (vi) a methanol dehydrogenase, aS-(hydroxymethyl)glutathione synthase, a glutathione-dependentformaldehyde dehydrogenase and a S-formylglutathione hydrolase; (vii) amethanol dehydrogenase, a glutathione-dependent formaldehydedehydrogenase and a S-formylglutathione hydrolase; (viii) a methanoldehydrogenase, a formaldehyde activating enzyme, amethylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolatecyclohydrolase and a formyltetrahydrofolate deformylase; or (ix) amethanol dehydrogenase, a formaldehyde activating enzyme, amethylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolatecyclohydrolase and a formyltetrahydrofolate synthetase.
 4. Thenon-naturally occurring microbial organism of claim 1, wherein themethanol metabolic pathway further comprises a formate dehydrogenase, aformate hydrogen lyase, or hydrogenase.
 5. The non-naturally occurringmicrobial organism of claim 1, wherein said non-naturally occurringmicrobial organism comprises two, three, four, five, six or sevenheterologous nucleic acids, each encoding a methanol metabolic pathwayenzyme.
 6. The non-naturally occurring microbial organism of claim 1,wherein said at least one exogenous nucleic acid encoding a methanolmetabolic pathway enzyme is a heterologous nucleic acid.
 7. Thenon-naturally occurring microbial organism of claim 1, furthercomprising one or more gene disruptions, wherein said one or more genedisruptions occur in one or more endogenous genes encoding protein(s) orenzyme(s) involved in native production of ethanol, glycerol, acetate,lactate, formate, CO₂, and/or amino acids by said microbial organism,and wherein said one or more gene disruptions confer increasedproduction of succinate in said microbial organism.
 8. The non-naturallyoccurring microbial organism of claim 7, wherein said protein(s) orenzyme(s) is a pyruvate decarboxylase, an ethanol dehydrogenase, aglycerol dehydrogenase, a glycerol-3-phosphatase, a glycerol-3-phosphatedehydrogenase, a lactate dehydrogenase, an acetate kinase, aphosphotransacetylase, a pyruvate oxidase, a pyruvate:quinoneoxidoreductase, a pyruvate formate lyase, an alcohol dehydrogenase, alactate dehydrogenase, a pyruvate dehydrogenase, a pyruvateformate-lyase-2-ketobutyrate formate-lyase, a pyruvate transporter, amonocarboxylate transporter, a NADH dehydrogenase, a cytochrome oxidase,a pyruvate kinase, or any combination thereof.
 9. The non-naturallyoccurring microbial organism of claim 1, wherein one or more endogenousenzymes involved in native production of ethanol, glycerol, acetate,lactate, formate, CO2 and/or amino acids by said microbial organism hasattenuated enzyme activity or expression levels.
 10. The non-naturallyoccurring microbial organism of claim 9, wherein said enzyme is apyruvate decarboxylase, an ethanol dehydrogenase, a glyceroldehydrogenase, a glycerol-3-phosphatase, a glycerol-3-phosphatedehydrogenase, a lactate dehydrogenase, an acetate kinase, aphosphotransacetylase, a pyruvate oxidase, a pyruvate:quinoneoxidoreductase, a pyruvate formate lyase, an alcohol dehydrogenase, alactate dehydrogenase, a pyruvate dehydrogenase, a pyruvateformate-lyase-2-ketobutyrate formate-lyase, a pyruvate transporter, amonocarboxylate transporter, a NADH dehydrogenase, a cytochrome oxidase,a pyruvate kinase, or any combination thereof.
 11. The non-naturallyoccurring microbial organism of claim 1, further comprising aformaldehyde assimilation pathway, wherein said non-naturally occurringmicrobial organism comprises at least one exogenous nucleic acidencoding a formaldehyde assimilation pathway enzyme expressed in asufficient amount to produce an intermediate of glycolysis and/or ametabolic pathway that can be used in the formation of biomass, andwherein said formaldehyde assimilation pathway comprises (i) ahexulose-6-phosphate synthase and a 6-phospho-3-hexuloisomerase, whereinoptionally the intermediate is a hexulose-6-phosphate, afructose-6-phosphate, or a combination thereof; or (ii) adihydroxyacetone synthase and a dihydroxyacetone-phosphate kinase,wherein optionally the intermediate is a dihydroxyacetone, adihydroxyacetone phosphate, or a combination thereof.
 12. Thenon-naturally occurring microbial organism of claim 11, wherein thenon-naturally occurring microbial organism comprises two exogenousnucleic acids, each encoding a formaldehyde assimilation pathway enzyme.13. The non-naturally occurring microbial organism of claim 1, whereinsaid non-naturally occurring microbial organism is in a substantiallyanaerobic culture medium.
 14. A method for producing succinate,comprising culturing the organism of claim 1 under conditions and for asufficient period of time to produce succinate wherein optionally theorganism is a Crabtree positive, eukaryotic organism, and wherein theorganism is cultured in a culture medium comprising glucose.
 15. Amethod of producing formaldehyde, comprising culturing the organism ofclaim 1 under conditions and for a sufficient period of time to produceformaldehyde, and optionally wherein the formaldehyde is consumed toprovide a reducing equivalent or to incorporate into succinate or targetproduct.
 16. A method of producing an intermediate of glycolysis and/oran intermediate of a metabolic pathway that can be used in the formationof biomass, comprising culturing the organism of claim 11 underconditions and for a sufficient period of time to produce theintermediate, and optionally wherein the intermediate is consumed toprovide a reducing equivalent or to incorporate into succinate or targetproduct.
 17. The non-naturally occurring microbial organism of claim 11,wherein at least one of said exogenous nucleic acids is a heterologousnucleic acid.
 18. The non-naturally occurring microbial organism ofclaim 12, wherein the two exogenous nucleic acids are heterologousnucleic acids, each encoding a formaldehyde assimilation pathway enzyme.19. The non-naturally occurring microorganism of claim 1, wherein saidnon-naturally occurring microbial organism is selected from the groupconsisting of: (i) Escherichia coli, Klebsiella oxytoca,Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes,Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis,Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis,Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor,Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonasputida; or (ii) Saccharomyces cerevisiae, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus,Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, and Rhizopusoryzae.